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Algal Turf Scrubber (ATS) Algae to Energy Project

Cleaning Rivers while Producing Biofuels and Agricultural and Health Products

Report to:

David Orr and Adam Lewis The Lewis Foundation % The Lipson Group Inc. 1422 Euclid Avenue, Suite 1500 Cleveland, OH 44115 January 31, 2010

Walter H. Adey Department of Botany National Museum of Natural History Smithsonian Institution Washington, D.C. 20560 Adeyw@si.edu

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Table of Contents

Executive Summary

Introduction

The ATS Energy Project

The Algae Primary Production 3-D Solution to the Diatom “problem” The ATS Ecosystem Nutrient Removal Oxygen Injection By-Products of ATS The All-Terrain ATS Issues to Consider

The Economics of ATS Operation

Plans for Project Expansion

References

Appendix

A. Algae of the Test Systems, Smithsonian Institution

B. Susquehanna Project, University of Maryland

C. Ozark Highlands Project, University of Arkansas

D. Biomass Processing, Western Michigan University

E. Project Extension (Chesapeake Algae Project, ChAP/Chesapeake Algae Consortium): organizations listed above, plus Blackrock Energy, StatoilHydro, Exelon Power, Constellation Energy, HydroMentia, Ecological Systems Technology University of William and Mary/VIMS,

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EXECUTIVE SUMMARY

Developed initially to control aquatic microcosms, Algal Turf Scrubbing (ATS) has a 30-year history of integration with the chemical and biological function of living ecosystems. Scaled-up, over the last 15 years, to several acre dimensions for tertiary sewage treatment, aquaculture and remediation of eutrophic creeks and canals, it has been used, coast to coast, in the southern U.S. The purpose of this investigation, funded by the Lewis Foundation, was: (1) to expand ATS technology to river scale, especially in more northerly climes; (2) to develop by- products from ATS-produced algal biomass, including biofuels; and (3) to develop the economics of large watershed application. To accomplish these tasks, several test ATS floways have been established on rivers, and numerous working associations with university scientific teams and companies have been developed.

The major finding of the field experimentation of this study on large non- point-source waters was a shift of ATS community structure from a standing crop dominance of filamentous green algae to that of filamentous diatoms. The biodiversity of the ATS algal communities was thereby greatly increased, enhancing the basic rationale of ATS. However, the lesser shear strength of diatom filaments, in the moderate energy environment of ATS, increased slough rate, and reduced expected productivity by 25-50% and nutrient removal by 10- 30%. This issue has been resolved with the development of a new type of basal, 3-D screen that retains diatoms, more than doubles algal productivity, and produces an entirely new field of potential process improvement. While broader use of this new technique is necessary, it is likely that further enhancement of nutrient remediation is now possible. Interface Global has joined our team, and is working to achieve mass production of these new screens; it seems likely that cost increases will be minimal as compared to process enhancement.

Photosynthesis produces oxygen, and the experiments of this study have emphasized that oxygen production by ATS is large; by manipulation of ATS flow to prevent excess supersaturation, injection of more than 35 tons/ATS acre/year into source waters should be possible. The more serious implication of water body eutrophication is hypoxia (“dead zones”), and in watershed amelioration, more emphasis needs to be placed on direct removal of hypoxia. This study produces an initial plan to accomplish this task for Chesapeake Bay.

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Aliquots of the algal biomass produced on the ATS floways of this study have been shipped to our associate team of chemists at Western Michigan University, where they have developed a physical/chemical separation process for carbohydrates, oils, proteins and minerals. The processed carbohydrate solutions were forwarded to our associate chemical engineering team at the University of Arkansas, where they have been converted to hydrogen and butanol using a membrane-modified, double Clostridium fermentation. The W. Michigan team has also demonstrated omega-3, PUFA’s in the ATS algal oils. While it seems likely that omega-3 separation will be more economic than the transesterification to biodiesel process that the Western Michigan team has also demonstrated, this should be determined at pilot scale. In plant growth tests, the residual proteins and minerals have been shown to be a quality fertilizer. However, the large quantity of silica diatom frustules, with a very large surface/volume ratio, in the mineral-rich fertilizer may additionally provide soil remediation that can lead to significant capturing and storing of atmospheric carbon. Both chemistry teams are now ready to begin expansion of this refinery process to pilot scale.

Previous large scale ATS systems have required time-consuming construction, earth-moving, grading and surface-preparation methods that have provided limitations to scale up. In this study, a new, at par cost, All-Terrain ATS unit has been developed that provides for rapid construction, dimensional flexibility, and applicability to unstable and difficult terrain such as landfills and river bottomland. This system will be used extensively in planned watershed scale-up.

With the development of an array of by-products, and the ability to work quickly and at large scale on rivers, tributaries and bays, this project has presented a preliminary economic plan for ATS utilization that can reduce the cost of both water remediation and algal by-product production. In the under way process of scale-up to pilot, and then full scale, for the Chesapeake Watershed, it will be necessary to further enlist environmental economists and modelers to guide and verify watershed-scale processes. However, managed by Blackrock Energy, and with the direct support of scientists from five universities, and companies such as Exelon Power, StatOil Hydro, Constellation Energy, HydroMentia and Ecological Systems Technology, this next step of The Chesapeake Algae Project, as the Chesapeake Consortium, seems well within reach.

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INTRODUCTION

Algal Turf Scrubbing (ATS) is a system for utilizing algal photosynthesis to control a wide variety of water quality parameters. Developed in the early 1980‟s at the Smithsonian Institution, as a biomimicry of coral reef primary productivity, ATS was initially used as a tool to manage an extensive series of living microcosm and mesocosm models of wild ecosystems. The models ranged from coral reefs to estuaries and fresh water streams; one of the largest of the mesocoms was a Chesapeake Bay system grading from tidal fresh waters to full coastal salinities. Applied to closed living models, ATS functioned to control nutrients, oxygen levels, carbonate systems, including calcification (through CO2 control) and to minimize toxic compounds from the local human-engineered environment. ATS also allowed the development of planktonic communities and planktonic borne reproduction in model ecosystems, as it has little effect on the planktonic component. This early development of ATS and the models it controlled are described by Adey and Loveland (2007). Those authors also describe how ATS techniques, scaled up, can restore our damaged rivers, bays and lakes.

Successfully scaled-up for nutrient removal, from point-source and semi point-source open waters, during the 1990‟s and early 21 st century, ATS use ranged from aquaculture and tertiary treatment of sewage to agricultural canal amelioration of nutrients. By 2009, eight scaled-up ATS systems had been built and operated from coast to coast, mostly in the southern tier of states. The northernmost unit was constructed on the lower eastern shore of Maryland.

Okeechobee, Florida, 2003

2.5 acre Algal Turf Scrubber (ATS)

processing 10-20 Mgpd of farm stream water

Scrubber (ATS) processing 10-20 Mgpd of farm stream water 7-acre ATS system Tilapia farm Falls City,
7-acre ATS system Tilapia farm Falls City, Texas Harvesting algae
7-acre ATS system
Tilapia farm
Falls City, Texas
Harvesting algae

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Two particularly successful early ATS operations were a 7 acre Tilapia operation in Falls City, Texas which produced commercial quantities of fish for nine years, and a ¼ acre, 250 K gpd tertiary sewage system in the northern Central Valley of California (Craggs et al, 1996). In the earlier ATS floways, considerable algal primary production was achieved (yearly means from 25-45 g(dry wgt)/m 2 /day), and in some cases the algal biomass was used as an animal feed and fertilizer. In these ATS systems, the algal source was local, mostly by self-seeding from source water, and the algal biodiversity was high, across the spectrum of algal groups. Filamentous green algae were dominant, in terms of produced biomass and nutrient uptake. During the last decade, the Ocala, Florida engineering firm HydroMentia built a variety of several acre dimension ATS systems for State/municipal water quality control of small non- point-source waters (agricultural canals, creeks), but had restricted its activities to Florida. A multiple-site 1440 acre ATS system had been designed to remove agricultural nutrients from the Suwannee River in northwestern Florida (Stewart, 2006), but at this time that project has not been funded beyond engineering design phase or implemented by the state government.

The purpose of this study, funded by a generous gift from the Lewis Foundation to the Smithsonian Institution, and described in this report, was four-fold: (1) to demonstrate non-point- source nutrient capture capability by ATS in stream to river scale environments with mostly lower nutrient concentrations than point-source waters; (2) to develop a system for expanding the utilization of produced algal biomass to biofuels and other by-products; (3) to develop a process for expanding the use of ATS capabilities to watershed scale, especially in the Chesapeake Bay Region, currently under both a Federal Executive Order and a Federal Court Order to solve nutrient/hypoxia problems; and (4) to demonstrate an economic basis for large-scale ATS non- point-source treatment, with a complex of by-products, in more northerly latitudes.

To accomplish these tasks and acquire the necessary associates and supporters, numerous ATS power point presentations were made at universities, NGO‟s and state and Federal government agencies throughout the Chesapeake Bay Watershed and at associated universities outside of the Watershed. Three university teams headed by Dr. Patrick Kangas at the University of Maryland, Dr. Marty Matlock at the University of Arkansas and Dr. John Miller at University of Western Michigan were inducted into the study; these scientific teams were

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provided Smithsonian subcontracts to demonstrate ATS capabilities, and to examine refining processes for the ATS algal biomass. The final reports of these partners are appended.

Associations were also formed with scientific teams at the University of William and Mary and its Virginia Institute of Marine Science (VIMS) on the York River on southern Chesapeake Bay, the Chemistry Department at the University of Arkansas (a butanol fermentation group headed by Prof. Jamie Hestekin), and several companies: Exelon Power, Blackrock Energy, HydroMentia, Statoil Hydro, Ecological Systems Technology, Constellation Energy and Interface Global. The contributions of these groups are discussed in this report along with several process diagrams to document their participation and the results of joint work. However, since they were not subcontract participants, funded by the Lewis Foundation gift, reports were not requested. Blackrock Energy, having brought together a coalition of additional universities and companies, is now serving as the lead organization in this consortium.

ATS floways were established on the Susquehanna, Great Wicomico and York Rivers in the Chesapeake Watershed and on the Springdale tributary of the Illinois River in Arkansas. Algal biomass from these systems and several of HydroMentia ATS units in Florida were sent to the University of W. Michigan for the study of its chemical composition and the development of separation processes. The extracted carbohydrates were sent to the Department of Chemical Engineering at the University of Arkansas and were processed to butanol and hydrogen using a semi-permeable membrane modification of the Ramey, Clostridium double-fermentation.

of the Ramey, Clostridium double-fermentation. Muddy Run, Susquehanna River - Three ATS floways –
of the Ramey, Clostridium double-fermentation. Muddy Run, Susquehanna River - Three ATS floways –

Muddy Run, Susquehanna River - Three ATS floways VIMS, York River

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THE ATS ENERGY PROJECT

The Algae

Previous work with ATS, from microcosm to moderate scale had concentrated on systems rich in either hard benthic environments (i.e. rock or branches), with abundant algal turfs as biofilms, or aquatic flowering plants that carried periphyton on their stems. Those ATS systems were highly dominated by filamentous green algae, particularly species of the genera Cladophora, Spirogyra, Microspora, Ulothrix and Rhizoclonium. Diatoms and cynobacteria were usually present, especially as epiphytes, but rarely provided significant biomass.

as epiphytes, but rarely provided significant biomass. Typical filamentous, green dominated algal turf, growing on
as epiphytes, but rarely provided significant biomass. Typical filamentous, green dominated algal turf, growing on

Typical filamentous, green dominated algal turf, growing on a basal plastic screen from a Florida ATS. Diatoms in the canopy in the diagram at the left; will form a brown tint later in cycle at right.

This investigation concentrated on river-stream systems, where either because of their large volume to bottom ratio and/or the predominance of sandy or muddy bottoms, planktonic algal communities, and those diatom communities specialized to living on sand and mud bottoms (rather than periphyton and algal turf communities), were dominant. While extensive seeding efforts from small, rocky periphyton-dominated local streams, with abundant filamentous green algae, were attempted, in most test floways, for most of the operational time, planktonic-sourced diatoms came to provide the majority of the biomass. The dominant species, by biomass, of

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these ATS floways were filamentous diatoms, particularly species of the genera Melosira, Fragilaria, Diatoma and Berkeleya. In species number, however, the unicellar diatoms, species of Nitzschia, Gomphonema and Navicula, were prominent. Investigations of the biodiversity and community structure of the algae on the floways of this study were carried out, and these species and genera, along with their abundances at each site are shown in Appendix I. In general, algal species biodiversities on these ATS systems were very high and this was especially true of the Susquehanna floways, where over 200 species of algae were tallied.

floways, where over 200 species of algae were tallied. Berkeleya rutilans Melosira nummoloides cf. Synedra sp.
floways, where over 200 species of algae were tallied. Berkeleya rutilans Melosira nummoloides cf. Synedra sp.

Berkeleya rutilans

where over 200 species of algae were tallied. Berkeleya rutilans Melosira nummoloides cf. Synedra sp. Several
where over 200 species of algae were tallied. Berkeleya rutilans Melosira nummoloides cf. Synedra sp. Several

Melosira nummoloides

where over 200 species of algae were tallied. Berkeleya rutilans Melosira nummoloides cf. Synedra sp. Several
where over 200 species of algae were tallied. Berkeleya rutilans Melosira nummoloides cf. Synedra sp. Several
cf. Synedra sp.
cf. Synedra sp.
Several diatoms
Several diatoms

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While some green algal species, filamentous and unicellular, were present on all ATS systems of this study, they were mostly minor elements of biomass. The filamentous green alga Spirogyra was moderately abundant on the Susquehanna floways during the summer, and the tube-former Ulva (Enteromorpha) was ubiquitous on the floways of estuarine rivers. Nevertheless, over time, we learned that the overwhelming biomass dominance of diatoms could not be significantly altered, in spite of considerable effort during the first six months, particularly at the Susquehanna site, to seed other species of periphyton. Similar efforts were undertaken for a shorter period in Arkansas. This was not necessarily a productivity concern, as diatoms, especially in temperate to arctic coastal waters, are the dominant producers of the plankton. Unfortunately, due to basic physiological and structural differences between diatoms and green algae, diatom filaments lack the tensile strength to consistently remain attached in the moderate energy environment of ATS systems, and this is a requirement of normal ATS function. Unlike in the earlier green algal-dominated ATS systems, significant slough, off the floway surfaces, occurred throughout the growing cycle (typically 7 days, though reduced to 5 days in mid- summer and increased to 14 days in mid winter).

ATS VIMS

May -July 2009 Cyanobacteria 2% Dinophyta 0% Berkeleya rutilans (43%) Melosira nummoloides (26%) Bacillariophyta
May -July 2009
Cyanobacteria
2%
Dinophyta
0%
Berkeleya rutilans
(43%)
Melosira nummoloides
(26%)
Bacillariophyta
Licmorpha sp complex
(7%)
Gyrosigma sp. (5%)
95%
Nitzschia sp. complex (5%)
Fragilariopsis sp. (3%)
Pseudonitzschia cf. multiseries
(2%)

Chlorophyta

3%

York River ATS: 95% of biomass is diatoms (Bacillariophyta

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Number of Species

CYANOBACTERIA

BACILLARIOPHYTA

ZYGNEMAPHYCEAE

CHLAMYDOPHYCEAE

CHLOROPHYCEAE

RAPHIDOPHYCEAE

CRYSOPHYCEAE

EUGLENOPHYCEAE

On the Muddy Run (Susquehanna) floways, algal slough was captured at the end of the floways with mesh bags, and was shown to be 10-20% of the harvested biomass. However, the mesh size of the bags had to be large (30um) to prevent blockage and flooding. Diatom filaments, made up of weakly attached cells, were prone to breakage, and subsequent passage of the smaller individual cells through the slough bags. Also, algae sloughed to the mesh bag ceased to efficiently photosynthesize, and add to system productivity. In addition, some algae likely died and were degraded by bacterial action, between harvests, in the slough mesh bags. Although ATS-function was otherwise normal, all of these processes reduced harvest. On the other hand, nutrient removal was only minimally effected, and this aspect of ATS operation will be discussed below. After several months operation on the Susquehanna floway at 2% slope, a second floway of 1% was added, side-by-side with the first unit in the hopes of reducing diatom slough. This second, lower-sloped floway had little effect on diatom dominance or retention, and algal productivity was lower. On the other hand, nutrient removal increased. This is probably a result of longer retention time, and greater organic particulate removal with the lower flow rate.

Muddy Run, Susquehanna River, Floways

Wood and Aluminium Floways species group comparison

2% slope (aluminum) & 1% slope (wood)

70

60

50

40

30

20

10

0

species group comparison 2% slope (aluminum) & 1% slope (wood) 70 60 50 40 30 20
species group comparison 2% slope (aluminum) & 1% slope (wood) 70 60 50 40 30 20
species group comparison 2% slope (aluminum) & 1% slope (wood) 70 60 50 40 30 20
species group comparison 2% slope (aluminum) & 1% slope (wood) 70 60 50 40 30 20
species group comparison 2% slope (aluminum) & 1% slope (wood) 70 60 50 40 30 20

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Wood Aluminium Total
Wood
Aluminium
Total

Primary Production

The high photosynthetic capability and primary production of algae is well known and has been the subject of the development of a potential new industry for algae to biofuels (Chisti, 2007; Ryan, 2009). However, most of the algae utilized in these new systems, whether photobioreactors or high-rate ponds are suspended, (floating or planktonic) in the water column; optimizing and harvesting that photosynthetic potential, economically, has proven difficult. ATS was developed as a simple, low cost means of achieving and utilizing algal photosynthetic and productivity potential using attached algae. When nutrients are moderately high and solar energy moderately abundant, the results of ATS are striking, and this has been repeatedly demonstrated in the southern tier of states from coast to coast. Numerous aquaculture and point-source or semi-point source treatment projects with productivities ranging from 25-45g(dry wgt)/m2/day, have been developed (Adey and Loveland, 2007; Mulbry et al, 2008; www.Hydromentia.com). While these rates of biomass productivity may not be as high as some photobioreactor methods, they are 5-10 items that of local agriculture. When used to clean wastewaters, as well as develop biomass products, the cost of the products, such as biofuels can be considerably lower than photobioreactor methods. Conversely, when ATS is used to produce algal by-products, the costs of nutrient removal from eutrophic waters can likewise be significantly reduced.

The ATS productivity initially achieved in this project, especially on the Susquehanna River floways (Table 1), where relatively low nutrients and annual solar energy were factors,

were less than expected.

units produced at 20-50% less than that of the seasonal expected values. As discussed above, this

was largely a result of the formation of a diatom-dominated community that tended to slough-off the floway, and in the case of greatest reduction that we further describe below, chironomid activity. The solution to this loss of productivity has been a relatively minor, low cost “tweakingof the ATS process, in part by increasing micro surface area in the growing screen. These new substrate screens, further discussed below, are expected to generally double production and nutrient removal on diatom-dominated systems and thereby bring non-point source treatment at least to the levels routinely achieved in point source amelioration.

While still about 3-4 times greater than local agriculture, test ATS

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Table 1

ATS

Floway

Comparison

summer ( 6-9 ) ------------------------------------this study------------------------------

 

High nutrients

Low nutrients

 

Site

Patterson, CA 1 (tertiary)

Springdale, AR (large sub urban creek)

Susquehanna, PA

Susquehanna, PA

S-154/Suwannee, FL. 2 (river)

(type)

2% slope-alumn.

1% slope-wood

length

area

153.8m

1100m 2

92.3m

30m 2

92.3m

30m 2

92.3m

30m 2

184.6m

5.76.106m2

flow rate

644l/min

77/l/min

50 l/min

44 l/m

12 Bl/day

 

mean Summer Solar (Gates, 1980)

590

 

546

(72.10 3 )

(63.4 10 3 )

 

540

Productivity

40

 

30.9

17.6

12.3

 

45

gdry)/m2/day

   

Incoming Nutrients

(mg/l)TN

5.0

 

9.68

1.38

1.38

 

1.08

(g/m2/d)NLd)

4.5

36

3:31

22:04

2.25

N/P

1.6 :1

62:1

11.5:1

11.5:1

10.1:1

mg/l TP

3.1

0.157

0.12

0.12

0.107

g/m2/d) PLd.

2.81

0.58

0.288

0.254

0.223

Nutrient Removal

           

%TN

22

 

30.5

28%

37%

 

44%

gN/m2/day

1.11

10.99

0.93

1.08

1.00

   

%TP

45

 

52.9

9%

17%

 

83%

gP/m2/day

0.73

0.31

0.026

0.043

0.185

Algal Composition

           

% diatoms % filament. Greens % cyanobacteria

23

 

76

77.5

77.5

 

(30) 3

69

20

14.5

14.5

(65.5)

7

4

6

6

(2)

     

1. Craggs et al, 1996

2. Stewart, 2006

3. Adey et al, 1993

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3-D solution to the diatom “problem”

About half-way through this investigation, it became clear that on larger muddier rivers, including estuarine “rivers”, a diatom community dominated by species of the filamentous genera Melosira, Berkeleya, Diatoma and Fragilaria would self-establish. Repeated seeding of periphytic green algae from smaller tributaries would alter this situation significantly only for a few weeks. The highly dominant diatom community of the rivers included filamentous species that could enter the benthic community of the ATS and “swamp” normal benthic species because of the overwhelming abundance of their cells and filaments in the continuously overflowing water.

As demonstrated earlier, the green-algae-dominated ATS community operating on a Florida canal, shows that similar diatom elements that occur on river ATS are also present (in that case different species of Melosira and Eunotia), but they do not displace the dominant green filaments. In those cases, the diatoms are epiphytic on the green filaments and become an

important (up to 30%), but not over-riding element of the algal turf.

“browning” of the growing biomass as it nears harvest, and the diatom filaments fill in the upper

structure of the algal “forest.”

Visually this is seen as a

The traditional substrate in the ATS system is a plastic screen. Many varieties of plastic screen have been employed, although a HDPE plastic of 3x5 mm mesh is typical, a wide range of mesh size has been used. On the scale of the ATS floway and the enhanced algal community these screens are 2-dimensional structures. The dominant diatom communities that occurred on river ATS systems quickly attach to these “standard” screens, but their filaments constantly “shear-off” in the moderate energy environment of an ATS, producing a lower standing crop and ultimately lower water remediation capabilities and by-product biomass. Diatom filaments have an entirely different structure from that of typical green algal filaments. In the latter case, the often massive cellulosic wall is continuous from cell to cell, usually with no break. Individual cells can die without compromising the integrity of the green filament, at least in the short term.

Diatoms, on the other hand, are basically cellular in construction, and have an entirely different kind of cell wall. The young, naked, diatom cell develops vesicles in its plasmolemma

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membrane, which in turn secrete amorphous silica within a matrix of protein, polysaccharide and lipid. When the rather complex silica frustule of typically four parts, two valves and two thin encircling girdles (holding the valves together) is completed, the entire complex is encased in a matrix of polysaccharide (sulphonated glucoromannan) (van den Hoek, 1995). The silica unit is the frustule; it is long-lasting, and after the death of the cell, it can become fossilized, sometimes as extensive deposits (diatomaceous earth). The frustules, and their individual cells are “glued” together by polysaccharides, often at spines, or held together in a polysaccharide matrix that can allow cell to cell sliding. Diatom unicells are often mobile, and can slide with a conveyor belt like movement of the matrix along grooves in the frustules. In some genera, such as Berkeleya, the “filament” is an extension of the polysaccharide sheath, in which the individual cells are randomly arrayed. Diatom cells can quickly attach to a substrate with the polysaccharide “glue” of their wall, and that is why they generally the first colonizers of new surfaces.

In conclusion, unlike green algal filaments (or red or brown algal filaments in sea water), diatom filaments are basically fragile and subject to breakage in the energy-rich ATS environment. To prevent this loss of diatom biomass, it was decided to develop a 3-dimensional screen to simulate the filamentous green algal structure that we could not maintain. Since in earlier systems, diatoms that attached to and entangled in the canopy of the dominant filamentous green algae remained on the floway, it seemed logical that if we supplied that structure artificially, we could more efficiently retain the diatoms on ATS floways.

A wide variety of off-the-shelf, deep pile throw rugs, with 1-2 cm thick loose fibers, were established on an experimental ATS system on the Chesapeake‟s Great Wicomico River (a 10-14 ppt environment at about mid-Bay, north to south). Also, several screens were specially woven, with 1-2 cm long Dacron fibers, to simulate the structure of the green alga “forest” that we could not consistently develop on river ATS units. The Dacron was employed because it would provide for minimal degradation under solar UV. Some of the rugs had a structure that considerably enhanced diatom retention. However, after several months of experimentation, the specially woven Dacron 3-dimensional (3-D) test screens provided the greatest productivity of all the test screens. These were established in the central part of the ATS test floway. Standard 2-dimensional (2-D) ATS screens were arrayed both above and below.

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Regular Screen
Regular Screen
Fully Developed Raised Filament
Fully Developed Raised Filament

Comparison of 2-D and 3-D screens prior to harvest

The 3-D screens consistently provided over 2 times the productivity of the standard 2-D screens (see below). Note that the plotted points are means of two 3-D and three 2-D screens; a t-test provides a high level of significance, p=0.0003. The fitted curves are sine curves anchored to expected peaks in June and December. This study is still underway, and as of late December, 2009, shows a production on 3-D screens of 2.7 x 2-D screens. The mean production for 3-D screens during December, the month of least incoming solar radiation, was 11.5 g/m2/day, as compared to 4.0 g/m2/day for the standard 2-D screen; this was twice the winter production on the Patterson tertiary ATS (Craggs et al, 1996) and about the same rate of production as standard 2-D screens in Florida during December (Adey et al, 1993).

same rate of production as standard 2-D screens in Florida during December (Adey et al, 1993).

2-D screens

same rate of production as standard 2-D screens in Florida during December (Adey et al, 1993).

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3-D screens

50.0 45.0 40.0 Great Wicomico River 35.0 30.0 2D vs 3-D Screens 25.0 20.0 15.0
50.0
45.0
40.0
Great Wicomico River
35.0
30.0
2D vs 3-D Screens
25.0
20.0
15.0
10.0
5.0
0.0
8/15/09
9/4/09
9/24/09
10/14/09
11/3/09
11/23/09
12/13/09
1/2/10

2-D20.0 15.0 10.0 5.0 0.0 8/15/09 9/4/09 9/24/09 10/14/09 11/3/09 11/23/09 12/13/09 1/2/10 3-D 2D Sine

3-D20.0 15.0 10.0 5.0 0.0 8/15/09 9/4/09 9/24/09 10/14/09 11/3/09 11/23/09 12/13/09 1/2/10 2-D 2D Sine

2D Sine25.0 20.0 15.0 10.0 5.0 0.0 8/15/09 9/4/09 9/24/09 10/14/09 11/3/09 11/23/09 12/13/09 1/2/10 2-D 3-D

3D Sine25.0 20.0 15.0 10.0 5.0 0.0 8/15/09 9/4/09 9/24/09 10/14/09 11/3/09 11/23/09 12/13/09 1/2/10 2-D 3-D

With the 3-D screen returning the structural environment of the standard complex, mini- forest algal turf, filament-sloughing is largely avoided and the expected productivity, based on previous small-stream, tertiary treatment and aquacultural systems, in the Chesapeake Bay area is more than recovered. It is likely that additional improvements in screen design will further increase filament retention and productivity. Interface Global carpet Company of La Grange, Georgia has been working with us to produce new, entirely synthetic, rug-like screens that are similar to our successful 3-D units. While several more iterations are probably necessary, it seems likely that the needed screen can be produced at large scale and low cost. Note that vacuum harvest is used in all of these studies, and the resulting biomass has been investigated to be certain that no synthetic fibers are present that might artificially increase harvest biomass.

There are likely many factors that are at work in the 3-D process. A major factor in productivity increase in photobioreactors is increase of surface area to limit solar self-shading by planktonic cells. That is a macro-approach that was seen in our early coral reef work (Adey and Steneck, 1985). In part, this current methodology is a “micro” approach of increasing surface area to provide additional attachment sites for diatom filaments. However, the 3-D process also creates “cells” or mini-volumes, a habitat in which water pulsing is likely modified to a swirling turbulence that increases mixing without filament shearing.

17

The ATS Ecosystem

Most algal to by-products production systems, including those producing biofuels, are monocultures of single algal species, and in many cases, the organisms employed are genetically-selected varieties. Genetically modified clones or GMO‟s (genetically modified organisms), have also been employed, and it is often suggested that the algal to biofuel industry will be successful only using species that have been modified to have specialized characteristics, such as oil production (Ryan, 2009). In production modes, these cultures are not bacteria-free, but they are at least as much monocultures as most agriculture crops, and require constant vigilance to prevent algal weeds and disease from overcoming the target algal species.

In a genetic context, an ATS floway is highly diverse with wild algal species. It is considerably more than a polyculture of several human-selected species, and perhaps is best termed a multi-culture of whatever algal species are available in the water body being treated. It is a culture, because the physical conditions are established by human action, the species best adapted to those conditions settle (as any wild ecosystem), and routine harvest takes place; inevitably some species are selected (by the conditions created) and others at least minimized. Only a single pair of ATS floways have been intensively monitored for algal diversity (the units at Muddy Run, on the Susquehanna River), and over 200 species of algae have been tabulated.

The typical ATS community additionally contains a diverse mixture of protozoans and small invertebrates, especially nematode worms and insects in fresh water and small crustaceans and polychaetes in estuarine waters. Such an ATS floway is an autotrophically-based ecosystem. However, by examining the percentage of nutrients removed as TN (total nitrogen) versus DN (dissolved nitrogen), the floway nutrient sink can be demonstrated to be typically 30-40% derived from particulates in the water column (including plankton). Thus, the ATS ecosystem clearly has a heterotrophic component. As earlier microcosm research had shown (Adey and Loveland, 2007), particulates are captured not generally by the filtering of plankton, but rather mostly by capture of organic particulates. Some of the protozoans and invertebrates on the ATS floway could be capable of mucous web capture of particulates, but two of the most abundant organisms on ATS systems, diatoms and blue-green algae, secrete abundant mucous to encase their cells, attach themselves to the substrate and , in the case of the diatoms, provide mobility.

18

Most diatoms can function as both autotrophs and heterotrophs (based in dissolved organics), and some can switch from one source to another (Graham and Wilcox, 2000). Although it has yet to be investigated, it is likely that there is an internal transfer of nutrients and energy from organic particulates through the bacteria, protozoan and invertebrate sub- community to diatoms, and this may be partly the source of apparently higher levels of diatom photosynthesis on ATS systems.

Nutrient Removal

Table 1 sums up the status of nutrient removal in the three primary floways of this study (see Appendix for full data). These are compared with previously published work, at high nutrient levels for the Patterson, CA tertiary treatment system (Craggs et al, 1996) and for the HydroMentia S-154 (2.5 acre) system in Florida, using as a proxy the engineered application to the Suwannee River (HydroMentia, 2005). The warm season data (June-Sept.) was employed because data were available for all compared systems for that time. Mid-winter data was absent due to winter system failure on the Susquehanna River; Springdale Creek (Arkansas) had a broken data stream and is being re-run during 2009-2010. The table is set up with the three diatom-dominated floways in the middle, the moderately high nutrient Patterson system on the left and the low nutrient Suwannee (S-154) on the right. The Patterson and Suwannee (S-154) floways are green algal dominated.

The extensive work of Mulbry and his associates (Mulbry et al, 2008) using ATS on agriculture wastes was considered for comparison in this table, since many of their conclusions support the earlier work at acre scales mentioned above. However, the Mulbry systems were recirculating, and low volumes made multiple passes over the ATS surface, whereas typical ATS remediation is single pass and involves large daily volumes. As a result, the Mulbry systems show high removal to loading ratios, although considering loading levels, nutrient removal is well within the range for single pass ATS. Although recirculating ATS is quite applicable to highly concentrated agricultural wastes, it would not be suitable for non-point source treatment. Where appropriate, this body of work is cited and discussed below.

19

The basic engineering design of ATS systems provides for a small, pulsing (or surging) water flow, on a very shallow floway, moving against the algal filaments which are held tight to growing screen on the “floor” of the floway. This low energy, oscillating water motion limits the capability of algal cells to deplete nutrients in the film of water immediately adjacent to each cell, thereby increasing nutrient uptake potential and primary productivity. Light flashing, due to alternate cell to cell shading and lighting in the pulsing water, also provides for a greater photosynthetic rate. The resulting higher photosynthesis and production rate (over “natural” periphyton and “micro-algae” situations) produces greater nutrient uptake rates both by metabolic (direct uptake) mechanisms and precipitation within cell walls for some nutrients, (e.g., P, Ca, Mg, Fe).

To some extent, at moderate to low concentrations, nutrient removal rates in ATS

systems are a function of nutrient concentrations in the body of water being treated. Mulbry et al (2008) and the engineering firm HydroMentia (www.HydroMentia.com) have demonstrated a direct relationship between nutrient loading (concentration X flow/unit area) and nutrient uptake for total nitrogen (TN) and total phosphorus (TP). At least at low to moderate concentrations of TN & TP, that uptake is directly proportional to nutrient loading. However, this relationship

does not extend to higher loadings where nutrients can become “saturating”.

(2008) have shown that nitrogen saturates in ATS algae at about 6.5% and phosphorus at about 0.9% (at low to moderate pH). In addition, earlier studies at very high nutrient levels showed that cyanobacteria can become dominating. Cyanobacteria are photosynthetic. However, many species can form extensive biofilms, and often are embedded in thick mucilage that limits the

mixing effects of the ATS system that act to break down the “stagnant” water adjacent to the algal cells. Thus, potential productivity and nutrient removal can be significantly reduced at high

levels of loading (Adey and Loveland, 2007).

considerable ability to produce nutrient depleted tissues (Adey, 1987). This is especially the case in ATS systems when nutrients in influent waters are unbalanced from the “ideal” N:P of approximately 16:1. Further complicating the generalized relationship between nutrient loading and uptake, algae have the capability to store nutrients when they are available in excess of

growth requirements (Mulbry et al, 2008). Finally, as shown in this report, restructuring the basal substrate (2D vs 3D), can radically change productivity and potential removal. Thus, at

20

Mulbry et al

Also, at low nutrient concentrations, algae have

this time, there is no “global”, fine-tuning model for nutrient removal in all ATS systems, although a specific system can generally be characterized within narrow parameters.

Available solar energy is the ultimate limiter of primary production in ATS systems, and where yearly data are available, a sine curve of both primary biomass production and nutrient removal can be produced (Craggs et al, 1996). Algal growth occurs throughout the 24-hour day and metabolic nutrient removal (eg, nitrogen) is independent of daily solar radiation. On the other hand, phosphorus removal (as well as Ca, Mg, Fe, etc.) can be dependent upon solar radiation/photosynthesis removal of CO 2 , followed by elevation of pH and the resultant precipitation within algal cell walls. Where pH levels can rise high enough, as in the example shown below for the Springdale Creek ATS system, such precipitation is directly proportional to solar energy capture on a diurnal basis. Algal community structure, in the physical sense, as demonstrated above, is also a determinant of primary production (and therefore nutrient removal). Nevertheless, in spite of wide day to day variability in any one system, removal follows productivity and tends to be consistent, varying seasonally. Given access to local wild communities of algae, algal production and nutrient removal in ATS systems is not significantly temperature dependent, as long as freezing of the water does not occur. This is consistently seen in production curves that follow light, even when temperature lags by several months.

pH

10.5

10

9.5

9

8.5

8

7.5

7

Springdale Arkansas Floway - pH
Springdale Arkansas Floway - pH

8/19/2009

8/20/2009

8/21/2009

8/22/2009

8/23/2009

8/24/2009

8/25/2009

Outflow

Inflow

Returning to Table 1 and several of the ATS systems utilized in this project, all three diatom rich sites are lower in biomass productivity than would be expected based on incoming

21

summer solar energy alone and the production rates achieved in Florida and California. The Springdale, Arkansas site is about 25% lower and the Chesapeake sites are about 40% lower. However, it is quite likely that utilization of 3-D screens on these systems, as demonstrated above, will achieve expected productivity or higher. The Springdale floway, can be compared to Patterson since it has a relatively high nitrogen loading. Here at 77% of the productivity of Patterson, the system produces a total nitrogen (TN) removal to load of 30.5%, 8.5% higher than Patterson‟s For total phosphorus (TP), the removal to load ratio is 53%, 8% higher than Patterson‟s. Thus, the utilization of 3-D screens to double biomass productivity is likely to make the Springdale floway one of the best ATS nutrient removal systems yet constructed.

In comparing the Susquehanna floways to that for the S-154/Suwannee design, summer production at the Susquehanna site was 50-60% less than that in southern Florida, even though in-coming summer solar radiation is about the same. Nevertheless, the total nitrogen removal rates are about the same as Suwannee and the removal to load ratio is about 25% lower. Doubling production with 3-D screening should bring Susquehanna summer removal rate considerably higher than Suwannee. For phosphorus, the removal rates are about 20% of Suwannee and the removal to load is 9 and 17% respectively of the Suwannee design. Doubling production with 3-D screens would still make this a poorer performer at its current length, although higher pH levels, with higher productivity, would improve performance. However, the Suwannee floways at 188m are twice the length of the Susquehanna floways, and the additional length is likely to significantly increase phosphorus removal due to precipitation. The mean available P in the summer Susquehanna River water is sufficient to provide a P removal rate that is slightly better than the Suwannee removal rate; thus the Susquehanna floway at twice the productivity (due to 3-D screens) and twice the length would likely be a superior performer for P removal. Although increasing system length is likely to reduce the efficiency of N removal, with doubled productivity it is still likely to be better than either Patterson or Suwannee.

All of these high diatom-dominated floways would have to be operated with 3-D substrate to be certain of performance potential. However it seems likely, given the information available, that diatoms on an ATS system can provide better nutrient removal per unit biomass than an ATS community that is dominantly green algae. Perhaps the diatom capability of

22

removing dissolved organics is a factor. More research is necessary, as greater understanding may further improve performance. Do the generally smaller diatom cells provide greater surface area for cell/water exchange in the turbulent environment of ATS? Do the silica frustules of diatoms become a lens or diffuser that improves light utilization in the light-flashing environment of ATS? Initially perceived as a problem, the dominance of diatoms on river scale ATS may open up considerably greater potential for water remediation and by-product development. Also, while conventional wisdom would maintain that nutrient removal is best done at or near critical point sources, this is not necessarily the case if management considerations and optimization of refinery production of by-products are considered. In addition, low to moderate concentration of nutrients are proportionally better utilized by ATS systems than higher concentrations.

Oxygen

Hypoxia as a result of excess nutrient-created algal blooms becomes a major factor of water quality degradation in rivers, lakes, and bays and even on very large bodies of water such as the Gulf of Mexico. Point source, bacterial-based nutrient treatment methods significantly aggravate this problem, especially if costly oxygen (air) injection is not widely employed. ATS injects a significant amount of pure oxygen into water bodies being treated. The amount of oxygen theoretically being injected is somewhat more than the tonnage of the dry organic biomass (ash-free) being produced, and even though it is not given a dollar value in traditional remediation, it could be the most important single factor in early stage clean-up. It can only be available using photosynthesis-based rather than heterotrophic bacterial-based waste-water treatment.

Diurnal studies of oxygen production were undertaken on both the Susquehanna and the Springdale ATS floways of this investigation. An example, over several days, from the Springdale study is shown below, followed by a summary of warm season diurnal oxygen production on the 2% (aluminum) floway on the Susquehanna River. The mean warm season collected algal production on this latter floway was 17.6 g(dry)/m2/day, and theoretically net oxygen production should be about 30% higher than organic (ash-free dry weight). Thus, with organic production (ash-free dry weight) about 53% of dry weight (see By-Products, below), net

23

production of oxygen should exceed 13 g(dry)/m2/day, although this analysis shows only 8.12

gO2/m2/day. However, when oxygen is allowed to supersaturate by several hundred percent, as

in this study, oxygen is being lost to the atmosphere; in this case, more than one third the amount

being produced is lost. This is not surprising, since routinely, on a sunny afternoon, in the algal

turf on an ATS floway, abundant oxygen bubbles can be seen. These bubbles tend to lift the algal

filaments up into the flow, being responsible, to some extent, for the tendency of the diatoms to

break free and join the slough. When oxygen injection is important, increased water flow can

avoid high levels of supersaturation and deliver maximum oxygen to the water column.

and deliver maximum oxygen to the water column. 300 Oxygen Saturation State, Springdale, Arkansas 250
300 Oxygen Saturation State, Springdale, Arkansas 250 200 150 100 50 0 8/19/2009 8/20/2009 8/21/2009
300
Oxygen Saturation State, Springdale,
Arkansas
250
200
150
100
50
0
8/19/2009
8/20/2009
8/21/2009
8/22/2009
8/23/2009
8/24/2009
8/25/2009
Outflow
Inflow
DO Saturation (%)
8/24/2009 8/25/2009 Outflow Inflow DO Saturation (%) Modeling studies relating ATS function to that of the

Modeling studies relating ATS function to that of the water body being treated, in

conjunction with pilot operation, are essential to optimizing the results of ATS treatment.

Hypoxic zones generally occur in deeper water and tend to be “locked in” by overlying warm

water in summer, although co-modeling can likely direct the solution of this problem.

Mean Daily Net Production 8.12 gO2/m2/day
Mean Daily Net Production
8.12 gO2/m2/day

24

By-products of ATS

During the 1990‟s and the early years of the 21st century, the algal biomass produced in ATS operation had been widely used (although largely experimentally) as an animal feed (fish, cattle and chickens) and as a fertilizer or soil amendment. Walter Mulbry and his team at USDA studied the role of ATS algal biomass as a slow release fertilizer; they also developed an effective system for ATS in-farm cleaning of waste nutrients from cattle waste, with the algae being used as a field fertilizer for corn (Mulbry et al, 2005).

With the increasing recognition that many valuable products could be obtained from algal culture, it became clear that the economics of water clean-up, using ATS, could be significantly improved with further by-product development. During the 1980‟s and 90‟s, the possibility of algal biofuels was examined by the DOE, National Renewable Energy Laboratory (Sheehan et al, 1998). Since that time many algal to energy projects have developed. Some algal to biofuels projects have concentrated on the production of biodiesel from oil-rich monocultures of planktonic or microalgae following an extensive search for strains with high levels of oil storage (Chisti, 2007). Much of this work relies on photobioreactors, and could only with difficulty be adapted to waste water treatment. Some effort has been undertaken to genetically modify algal strains, using recombinant DNA to combine desirable characteristics from one or more algal strains into the “ideal” species. This has the same basic problems of escape to the wild and dilution of natural biodiversity as agricultural modification. However, with algal to biofuel methods, such a process would be pushing the envelope at both ends of the scale, very large area and microorganisms.

A very different approach, using high rate ponds (raceways with circulating paddle- wheels), converts algal biomass to methane gas, and has been applied to polluted farm run-off in southern California. Also, as noted above for diatoms, some algae have the capability to ingest both soluble and particulate organics, heterotrophically, independent of their normal mode of photosynthesis. However, this requires an independent source of organics, presumably waste biomass. A recent review of the field by the NRDC, Cultivating green Energy, The Promise of Algae Biofuels” (Ryan, 2009) provides a critical examination of the processes currently underway to convert algal biomass to energy. Since the result of most algal production is a

25

biomass with high water content, an energy/carbohydrate conversion requiring drying before refining makes little life-cycle sense. Thus, fermentation is the most likely first step in a conversion process.

The production of ethanol by yeast fermentation of plant sugars has become the dominant liquid biofuel in the U.S. However, ethanol carries low energy per liter (73% of gasoline), is a strong solvent, and is highly volatile, characteristics requiring special handling. It is processed as a low percentage mixture with gasoline. An alternate fermentation, ABE, using Clostridium bacteria, was developed during the first World War, mostly for acetone, the butanol component serving as an industrial solvent. ABE was the primary source of butanol as an industrial solvent until mid 20 th century, when conversion from crude oil refining began. The four carbon alcohol butanol, can function as a direct 1:1 replacement for gasoline, and has 96% of the energy per liter of gasoline. It is also six times less evaporative than ethanol, is less corrosive, less susceptible to water contamination, and produces less CO2 per BTU of energy supplied

This century-old fermentation, using a variety of carbohydrates, had been greatly improved during the 1990‟s by David Ramey (Ramey and Shang-Tian Yang, 2004; www.butanol.com) using two separate, sequential cultures of Clostridium tyrobutyricum and Clostridium acetobutylicum with hydrogen and butyric acid produced in the first stage and butanol, from the butyric acid, in the second. Nevertheless, the process had remained somewhat unstable, and with an improved but still modest efficiency.

More recently, Dr. Jamie Hestekin, a semi-permeable membrane specialist, had established a laboratory, at the University of Arkansas, funded to use membranes to improve the Ramey butanol fermentation. Since his R&D process was nearing successful completion, using corn sugars, the decision was made in this project to concentrate on butanol as the algal biofuel, and to look for other by-products from the relatively small quantities of residual oils. The primary question we had to answer was: would the algal sugars, and other associated carbohydrates, be different enough from the standard corn sugars to upset the Clostridium tyrobutyricum in the front stage of the modified Ramey process?

26

The Ramey Process, a dual, two- species, Clostridium fermentation
The Ramey Process, a dual, two-
species, Clostridium fermentation

Modified two -step Ramey process

utilized at the University of Arkansas

a dual, two- species, Clostridium fermentation Modified two -step Ramey process utilized at the University of

27

Algal biomass from all of the primary ATS test sites, except Springdale, Arkansas, and from several of the HydroMentia ATS units in Florida, especially an estuarine unit on the Caloosahatchee River draining to the Gulf of Mexico Coast, was used to develop a bench scale separation process. Dr. John Miller, and his associates at the University of W. Michigan, first devised a pattern of physical/chemical separation for the diatom-rich, but complex algal biomass from the ATS units, as shown in the following diagram. Although some of the carbohydrate solution was processed to ethanol, through several procedures as described in the University of W. Michigan report in the appendix, as an economic process this approach remains inconclusive at this time.

Algal Composition & Biofuel products

at this time. Algal Composition & Biofuel products Co-Products • High N, P • High Silica
at this time. Algal Composition & Biofuel products Co-Products • High N, P • High Silica

Co-Products

High N, P

High Silica

Butanol / Ethanol
Butanol /
Ethanol
Biodiesel PUFAs
Biodiesel
PUFAs

9 Nov 2009

13

Several batches of the algal-derived carbohydrate solutions were forwarded to the Hestekin Laboratory at the University of Arkansas and were successfully processed to hydrogen and butanol. The advantage of the Clostridium fermentation in the Hestekin process is its general lack of sensitivity. We now know that the bacteria can accept both soluble algal sugars as well as the insoluble carbohydrate components. Although basically anaerobic, the fermentation is not upset by small quantities of oxygen, making it relatively easy to operate as an industrial process.

28

According to the estimates by the W. Michigan Laboratory, 36 gallons of butanol per/dry ton of algae would be produced from the ATS algal feedstock. At approximately 30g(dry wgt.)/m 2 /day (48 tons/acre of ATS/year), as discussed above, this would yield 1700 gallons of butanol per acre per year. If all of the oils present in the algal biomass were to be converted into biodiesel, a relatively simple transesterification process, also described in the W. Michigan report, an additional 800 gallons of biodiesel could be produced. However, approximately 20% of total oils are composed of high value Poly Unsaturated Fatty Acids, (omega-3 oils). As marketed retail today omega-3 oils are typically at a concentration of about 25%, and it seems likely that a mature processing operation would produce either high value Omega-3 or biodiesel fuel. In the economic analysis that follows, omega-3 production is shown as the more likely process. The residual oils excess to omega-3 production could be transesterified to biodiesel or converted to some other products. Omega 3‟s are currently obtained from keystone fish that are not being sustainably harvested, so this direct algal source could be environmentally crucial.

Algal Biomass Pre-

processing

Percentages indicate relative amounts of dry algal biomass after each step
Percentages indicate
relative amounts of dry algal
biomass after each step

9 Nov 2009

14

29

In the NREL investigation of micro algae for biofuels (Sheehan et al, 1998), the target was oils, particularly for transesterification to biodiesel. Some oil-rich diatom species were included in that work. However, as seen in the work of John Miller at al. in this study, most diatoms have low oil content. On the other hand, as noted above, diatom cell walls are rich in easily-reduced polysaccharides that appear tol be acceptable to Clostridium tyrobutyricum without prior enzyme digestion. Cellulose appeared in these algal turf assays, but may have derived from the associated green algal filaments; the algal turf biochemistry of this study included some samples from Florida ATS units in which green algal filaments were likely the dominant algae. Initially seen as valuable in monoculture for oil-based biofuels, natural diatom cultures are apparently best-suited for butanol fermentation, with the oils having modest omega-3 content of high value for health food processing.

Finally, roughly 22 (dry) tons per acre per year of nutrients, minerals and residual proteins are left after the extraction of the higher value by-products. As the W. Michigan study has demonstrated (see Appendix D), and as shown by Mulbry et al (2005), this can be used as a high quality fertilizer. As shown below, the abundant silica diatom frustules are highly ornamented, and possess abundant pores or areolae. Diatom frustules are typically on the scales of 10‟s of a um in dimension, and their spines, ridges and areolae on a um scale. However, it has also been demonstrated that the frustules have a porous and “cellular” nanostructure at the nm scale that provides virtually infinite silica surface (Crawford et al, 2009). If properly processed so that the silica diatom frustules remain, the very high surface area of this “ash” component would likely provide a soil amendment that increases tilth, water and ion retention. This in turn, under an appropriate cultivation routine, could provide increased organic buildup, improved agricultural production and, at large scale, removal of significant carbon from the atmosphere. Soil studies are necessary to determine whether this route to carbon sequestration is viable.

The pre-processing procedure, as shown here, would probably have to be modified to bring forward the polysaccharide enzymes, perhaps with mild ultrasound treatment, before mechanical chopping. The silica frustules being relatively dense could be removed with centrifugation and then the organic residue treated with ultrasound sufficient to break down the

30

plasmolemma and release cells contents for continued processing. At the end of the refining

process, the silica frustules could be re-mixed with the residual nutrients, minerals and proteins

to produce the high grade fertilizer discussed above. Breaking down cell walls to get at the cell

contents is a typically difficult early step of producing a biofuel. It is especially difficult in

higher plants where the cellulose is usually infiltrated with the highly resistant lignin. In diatoms,

the silica cell wall is embedded in polysaccharides, relatively easily degraded and usable

compounds.

relatively easily degraded and usable compounds. Residue of a portion of algal turf from a Muddy

Residue of a portion of algal turf from a Muddy Run floway. Treated to remove organic materials and examined under the Scanning Electron Microscope (SEM), the abundant ornamentation and virtually infinite surface area of the complex silica frustules of diatoms, is apparent. Kept intact in processing, these frustules will greatly increase the fine porosity of soils in which the ATS refining residue of proteins, nutrients and minerals is employed as a fertilizer.

31

The All-Terrain ATS

The Algal Turf Scrubber process requires modest land area and often is criticized for this characteristic. However, as Table 2 shows, 15 times as much biofuel per unit area can be produced from ATS than from corn. At least in agricultural regions where corn is grown for ethanol, or soy beans for biodiesel, it makes little sense to argue that ATS requires too much land. Later in this report, an ATS model for Chesapeake Bay will be described which will provide quite sufficient land for Bay recovery. Nevertheless, there are situations in

urban/suburban regions where land would be difficult to obtain.

sites along rivers in rural areas where instability of substrate or irregularity of terrain would make standard ATS units impractical. While small temporary test ATS units can be established to discover the basic parameters that can lead to an efficient full scale system, this can be very time consuming. The larger scale units developed and utilized over the last decade require extensive grading and surface preparation as well as considerable concrete construction. Once committed, it is very difficult to alter these systems to new or undiscovered conditions.

Also, there are otherwise ideal

Algae vs. Corn Biofuel potential

FermentationBiodiesel Biomass yield % Carbohydrate Carbohydrates available % fermented Carbohydrates used

FermentationBiodiesel

Biomass yield

% Carbohydrate

Carbohydrates available

% fermented

Carbohydrates used

Fermentation efficiency (C into fuel)

Fuel produced

potential ethanol production

potential butanol production

triglycerides

Triglycerides available

conversion efficiency

Triglycerides used

Potential Biodiesel production

Triglycerides used Potential Biodiesel production ATS Algae Corn 50 3.1 dry ton/acre/yr 25%
ATS Algae Corn 50 3.1 dry ton/acre/yr 25% 35% 12.5 1.1 ton/acre/yr 90% 90% 11.3
ATS Algae Corn 50 3.1 dry ton/acre/yr 25% 35% 12.5 1.1 ton/acre/yr 90% 90% 11.3

ATS Algae

Corn

50

3.1

dry ton/acre/yr

25%

35%

12.5

1.1

ton/acre/yr

90%

90%

11.3

1.0

ton/acre/yr

51%

51%

=m alcohol /(m alcohol +M 2CO2 )

5.75

0.5

ton/acre/yr

1822

159

gal/acre/year

1775

155

gal/acre/year

6%

3

ton/acre/yr

95%

2.9

ton/acre/yr

810

gal/acre/year

1775 155 gal/acre/year 6% 3 ton/acre/yr 95% 2.9 ton/acre/yr 810 gal/acre/year
1775 155 gal/acre/year 6% 3 ton/acre/yr 95% 2.9 ton/acre/yr 810 gal/acre/year
1775 155 gal/acre/year 6% 3 ton/acre/yr 95% 2.9 ton/acre/yr 810 gal/acre/year
1775 155 gal/acre/year 6% 3 ton/acre/yr 95% 2.9 ton/acre/yr 810 gal/acre/year

Algae Yields are estimates from Dr. Walter Adey.

Algal carbohydrate compositions from Dr. John Miller (WMU) measurements of ATS samples

Corn yields are from the US Dept. of Energy Billion - Ton Vision .)

32

The All-Terrain ATS was developed specifically to utilize the otherwise waste area of capped landfills. However, because of the ease and rapidity of All-Terrain ATS construction, its flexibility for expansion in width or length, the potential for rapid re-siting, at cost at par with standard ATS units, this system opens up land areas that would otherwise be unavailable for ATS water remediation. An additional value of the All-Terrain systems is that it allows for the efficient injection of stack gas CO 2 both to improve productivity as well as provide for additional CO 2 removal from the atmosphere. While Mulbry et al (2008) were not able to demonstrate CO 2 enhancement of productivity and nutrient removal in their re-circulating systems, their dairy water nutrients were added one time in the morning and diurnal effects were not studied. It is likely that nutrient availability and carbon availability did not coincide in the experiments. Since CO 2 addition has been routinely used successfully in photobioreactor production (Chisti, 2007), it is possible that this was a experimental flaw in the Mulbry et al study.

that this was a experimental flaw in the Mulbry et al study. All-Terrain ATS of one

Issues to Consider

The ATS process of producing algae for water quality remediation, while providing an array of bi-products, including biofuels, has been remarkably free of significant problems; a few require mention.

The ATS process has been developed, in part tuned to avoiding diseases and the very few invertebrate pests (grazers) which could inhabit biomass production and nutrient removal. Especially on river systems, where the algal biodiversity of ATS units is particularly high, significant disease is highly unlikely. Viral or bacterial invasion would likely just shift community structure, with unaffected species taking over dominance. On the other hand, a limited array of algal invertebrate grazers can be present. Amphipods are small, shrimp-like arthropods that have many types of feeding behaviors. Some of the commonest species in benthic environments are grazers of algae. Data taken on the VIMS floway shows that biomass build-up and grazing amphipod abundance are closely related. When this relationship is considered, and harvest schedules closely adhered to, amphipod grazing has never been a significant issue in marine or estuarine ATS systems.

VIMS Algal Floway: Optimal harvest schedule

VIMS Algal Floway: Optimal harvest schedule

VIMS Algal Floway: Optimal harvest schedule
VIMS Algal Floway: Optimal harvest schedule Emmett Duffy and Elizabeth Canuel, VIMS
VIMS Algal Floway: Optimal harvest schedule Emmett Duffy and Elizabeth Canuel, VIMS

Emmett Duffy and Elizabeth Canuel, VIMS

VIMS Algal Floway: Optimal harvest schedule Emmett Duffy and Elizabeth Canuel, VIMS
VIMS Algal Floway: Optimal harvest schedule Emmett Duffy and Elizabeth Canuel, VIMS
VIMS Algal Floway: Optimal harvest schedule Emmett Duffy and Elizabeth Canuel, VIMS
Algal accumulation Daily production 2 4 6 Grazer invasion
Algal accumulation
Daily production
2
4
6
Grazer invasion

Experiment

Plots harvested daily for 10 days

measured algal production, grazers

Results

Algae produce rapidly

Pests invade more slowly

Optimal harvest at ~6 days (summer)

rapidly Pests invade more slowly Optimal harvest at ~6 days (summer) 1 October2009 ChAP Project Kick
1 October2009 ChAP Project Kick -off meeting
1 October2009
ChAP Project Kick -off meeting

34

In freshwaters, amphipod grazing is replaced by a single group of insects, the Midges. The non-biting, non-eating flying stages are reproductive phases that lay eggs in shallow water and on wet surfaces. The resulting larvae, called chironomids, are grazers of algae that grow in those waters and are potentially “goat-like” in greatly reducing algal productivity potential. During the past 20 years, in 14 out-door ATS floways in which chironomids could have been a problem, they have had a known presence of note in only four units. In two of those (the Patterson and Springdale systems) they occurred only periodically and were easily controlled by close observation and early harvests. At one tertiary treatment unit on the eastern shore of Maryland, where the ATS unit was unfortunately located adjacent to a sewage plant skimmer with a continuous production of Midges, chironomid infestation was serious. This case was controlled by Bacillus thuringiensis a bacterial addition inimical to the larvae; as a result, successful operation was achieved.

During the second summer of operation of the Muddy Run Susquehanna floways, chironomid infestation was serious (virtually none appeared the first summer). While this reduced biomass production considerably, it apparently had minimum effect on nutrient removal. Presumably, the chironomid larvae consumed a considerable amount of algae, reducing biomass (potentially available energy as biofuel), but retained a large portion of the nutrients which were removed with the larvae on harvest. Unfortunately, this site was remote from the University of Maryland base of operations, and adequate “short harvest” methods could not be routinely applied. This situation was also highly unique in that the larvae apparently arrived with the water flow from the stored water reservoir. The stored water reservoir emptied daily, producing large areas of mud flats, perhaps uniquely suited for both Midge egg-laying and larval release. That the infestation occurred in one year and not another, might be related to weather conditions. In general, chironomid grazing in ATS freshwater systems can be viewed as a potentially periodic, but usually a quite controllable issue. It has never been a problem on the larger Florida ATS units or on the large aquaculture unit in south Texas. On the other hand, those systems tend to be characterized by flocks of small, wading birds, which likely feed on any invertebrates present in the ATS. No large ATS system, of an acre or more, has suffered significant chironomid infection.

35

Considering the typical mode of grazing used by chironomid larvae, it seems likely that 3- D screens will reduce their effectiveness. The larvae construct small mucous/detritus “tunnels” from which they foray out to graze. It is unlikely that this can be accomplished on the raised filaments, and, as a result, they would be restricted to the basal part of the screen. This issue needs to be examined further, but it is likely that a screen can be devised that will exclude chironomid larvae from effective grazing.

A single floway in Florida has been struck by periodic algaecides; these were likely herbicides used on orange groves and flushed into the creek being treated during heavy rains. While potentially a serious problem in treating small creeks, dilution would likely prevent this problem on large rivers. Nevertheless, the poisoning of algae at tributary scale should be regarded as a more serious problem than eutrophication, since algae are at the base of most aquatic food chains. If algae cannot be grown due to anthropogenic toxins, then we have created “dead water swimming poolsout of our natural water. In this regard, ATS systems can be regarded as a water canary, capable of warning that waters are unacceptably toxic. ATS can function in this way also by degradation or accumulation of low level industrial and agricultural toxins.

Two of the ATS systems in this project (Susquehanna and Springdale) were shut down by freezing for about two months each. Freezing of the water in these systems occurred, in spite of considerable efforts to keep them running. Neither the Great Wicomico nor the York River raised floways, at mid and lower Chesapeake Bay respectively, were seriously affected by the hard and prolonged cold-wave of early January, 2010. Up to 40 o N latitude on each coast and

35 o N in mid country, properly designed and operated systems should be able to overcome this

problem. Nevertheless, as briefly described above, the co-location of large scale ATS with fossil fuel power plants can allow ice-free winter operation (using waste heated water effluent from the power plants) as well as to provide for significant CO 2 recycling.

36

THE ECONOMICS OF ATS OPERATION

Since well before the Green Revolution, the potential for algal culture systems to produce valuable products has not been in doubt. However, the cost to do so, in competition with agricultural products, most of which are grown on land cleared from forest centuries ago, has been a serious obstacle to the advancement of algal systems. Unfortunately, the full costs of agriculture, including soil and nutrient loss to natural waterways, are seldom included in agricultural economic analyses, so that a true comparison is difficult. In addition, in parallel with human experience over millennia of agricultural development, monocultural algal solutions, and more recently genetically modified solutions, have been sought. There is little question that this can be done, since algal isolates have been cultured in the laboratory for many decades. However, the complexity of achieving agricultural levels and methods of algal production over decades, when the processes for higher plants took millennia, provides a serious economic test.

The ATS system has overcome most of the large scale algal culture problems by using algal “weeds” that are available, year-round in the waters being treated. Moreover in moving to non-point-source treatment, it has become clear that an enormous diversity of “weedy” species is readily available in natural environments, and disease and competition are not issues of concern. Pests and excessive chemical pollution provide minor concerns, as we noted above. However, the economics of constructed systems as opposed to open field agriculture remains the primary issue in the production of algal products. As shown below, if value is achieved for water clean- up, ATS provides a very cogent economic scenario.

Table 3 presents an economic model for treatment of the Susquehanna River at or near its confluence with Chesapeake Bay. The 3000 acre model is employed because at this dimension ATS will remove the amount of phosphorus independently assessed as necessary to return the upper Bay to nutrient health; at this dimension, ATS will also inject enough oxygen into the water column to securely remove the hypoxia (dead) zone that has come to characterize the upper Bay. In addition, an engineering design, with economic analysis, for the Suwannee River in Florida has been completed by Hydromentia and provides the basic accounting to develop such a large scale model.

37

Chesapeake Bay Journal, November, 2009

Karl Blankenship. Report released for state of

Cheasapeake Bay and it’s rivers”

Required to Reach Target:

Chesapeake Bay P reduction 1.45Mlbs/year

Susquehanna River P reduction 1.04 Mlbs/year

3000 acres of ATS on Susquehanna Effluent

will remove: 1.5Mlbs of P; 9Mlbs of N/yr

will potentially add: over 200 Mlbs of oxygen/yr

will potentially increase upper Bay oxygen

concentration by 7 mg/l, thus removing hypoxia

Currently efforts are underway to join the stack gas CO 2 of a Constellation coal-fired power plant, as well as warm water effluent to ease winter operation, with the more-than- adequate area potentially available at the Aberdeen Proving Ground, at the confluence of the Susquehanna River and the Chesapeake Bay. This base is presently being reviewed for base closure. Whether or not this action is completed, there is a large area on this base that could be considered for co-use. The economic model presented in Table 3, is based on this situation, although other sites are also possible.

As the economic model demonstrates, given a nutrient trading or banking scenario, cost is not a significant factor in Bay clean-up. Indeed, it is likely that utilized in this manner, ATS clean-up of Chesapeake Bay is a green economic engine. Similar operations could be developed on the Potomac and James Rivers, potentially using available military land. This would not only provide a return of Chesapeake Bay to health, but would provide an economic boost to the Bay in greatly improving the fisheries of these waters that have been so long under environmental stress.

Table 3 provides an economic analysis based on a set of ATS units of 3000 acres total near the mouth of the Susquehanna River. Costs are derived from the Suwannee River (1440 acres) engineering design, with a 20% discount based on new technology and scale up factors. Nutrient values are Chesapeake Bay Commission (CBC) published mean nutrient removal costs

38

from 2004. Based on current anecdotal information this provides about a 27% discount on the current values (approximately $200/kg P $10/kgN). However, as we shall show, allowing for the value of by-products, ATS systems operated by a public, non-profit entity solely to reduce nutrient removal costs, could remove nutrients (and carbon) at about 50% of current costs.

The analysis of Table 3 is written to be carried out by a quasi government agency, with 30-year construction bonds at 5%. Carried out by a private engineering firm the difference between ATS nutrient removal costs and the market price, minus the cost of capital, would have to be negotiated. Given a functioning nutrient banking system, a significant return on investment could be available to drive clean-up efforts.

39

Table 3

Economic Analysis

Upper Chesapeake Watershed Project

3000 Acres Based on Suwannee River ATS Design

ATS Production: 30g(dry m 2 /day (48 tons/ac/yr)

Banked Credits for Nutrient Removal (Biomass Prod . 144,000 tons/year)

(Chesapeake Bay Comm., 2004; discount approx. 27% current cost)

Nitrogen (3%) 4320 tons @ $3.86/lb. (CBC $8.50/kg)

$33.4M

Phosphorus (0.4%) 576 tons @ $66 /lb. ( $145.20/kg)

76.0M

Carbon (38%) 54,720 tons @ $ 20/ton

1.1 M

Sale Returns on By - Products

$110.5M

Biofuel (butanol) 36 gals./dry ton, 5.2M gals. @$2.50/gal

$13.0M

Fertilizer (est. 54% of biomass) 77.8K tons @ $200/ton

15.6M

Hydrogen 2 M lbs @ $2.10/lb (energy equiv.)

4.1M

Omega-3 oils, 3.4 gals./dry ton, 490K gals @$100 gals. whsl.

49.0M

(Biodiesel, optional 2.5M gals. At $2.50/gal; 15.6M )

Costs

Gross Return: $188.1M

$ 81.7M

3000 acre ATS, upper Chesapeake Bay

Capital (5%) 30 yrs. plus O&M , incl. land Suwan. Proj. 06

$127 M

Refining (all products, est. 20% of product value)

$15.5M

Administration (10% of capital cost + O&M cost)

$12.7M

Profit or Re-investment

$32.9M

Gross Cost: $188.1 M

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The refining process for by-products needs to be expanded to pilot scale and then financially re-analyzed. Also, life cycle analysis needs to be carried out, especially for the refining process. It seems likely that the hydrogen produced in butanol fermentation would be most efficiently utilized on site to provide power for refinery operation. Also, CO 2 injection into ATS systems has not been performed at large scale, although the technology exists to do so using the All Terrain system, and application does not appear to be difficult. This aspect of project economics would have to be linked to carbon trading. Rivers in the Chesapeake Watershed have moderate alkalinity and in-water CO 2 supply is not a serious problem. The considerable rise in the pH of test systems shows that CO 2 stress is being placed on productivity. CO 2 injection, spread-out on floways can significantly increase algal productivity. On the other hand, this will delay phosphorus precipitation down the floway. These two opposing needs will have to be worked out in pilot operations. ATS has already been constructed and routinely operated for several years at multi-acre scale at a half-dozen sites. It is certainly time to expand to a 25-50 acre dimension pilot with a similarly scaled-up refining plant.

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PLANS FOR PROJECT EXPANSION

Now that it has been established that ATS remediation (nutrient removal and oxygen injection) is technically and economically feasible for non-point source waters (rivers and bays) at least up to mid latitudes, it is essential that this endeavor expand to multi-acre pilot demonstration scale. Since the Chesapeake Bay has demonstrated significant environmental problems, and the Federal Government has committed itself to solving this long-standing issue, it is here that our efforts will be concentrated.

A coalition of interested parties, convened and managed by Blackrock Energy, including the partner universities of this study plus the Universities of Virginia and Penn State University and the energy companies Exelon and Constellation Energy, have indicated a strong interest in forming a consortium to move ATS to pilot scale. This coalition has developed a project for both water-cleaning and by-products, including butanol, on the upper Chesapeake Bay. The Constellation coal-fired plant adjacent to the Army‟s Aberdeen Proving Ground will provide the land, heated effluent water and stack gases from its plant to expand to the next phase. Scientists at the Universities of W. Michigan and Arkansas are prepared to ramp up over one year, first to algae shipped “by the barrel” and then to a mobile refinery hauled to the Constellation site by year‟s end.

Exelon and Constellation plan to work together to support this endeavor with the goal of sourcing both funding and the necessary political support. Once fully successful at pilot scale (1- 3 acres), the intent is to expand to a 25-50 acre industrial pilot and within a few years to full acreage necessary for upper Bay clean-up. Tentatively, land area on the Aberdeen Proving Ground is being considered for this scale up. Additional efforts will also be initiated to develop similar facilities on the Potomac and James Rivers, thus looking to ameliorate the entire Bay as well as to provide significant quantities of by-products, including biofuels. A series of meetings are planned by all parties during early 2010 to bring this effort to fruition.

A recent proposal, developed by Blackrock Energy to the U. S. Department of Energy, in support of the Algae Consortium effort, is included in the Appendix. Constellation Energy and Exelon Corporation are the lead organizations in this proposal.

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REFERENCES

Adey, W. 1987. Food production in low-nutrient seas. BioScience 37: 340-348.

Adey, W. and K. Loveland. 2007. Dynamic Aquaria: Building and Restoring Living ecosystems.

3 rd edition. Elsevier, Amsterdam. 508 pp.

Adey, W, C. Luckett, and K. Jensen. 1993. Phosphorus removal from natural waters using

controlled algal production. Restoration Ecology 1: 29 39.

Adey, W. and R. Steneck. 1985. Highly productive eastern Caribbean reefs; synergistic effects

of biological, chemical, physical and geological factors. NOAA Symposium Ser. For

Undersea Research 3: 163-187.

Chisti, Y. 2007. Biodiesel from microalgae. Biotechnology Advances 25: 294-306.

Craggs, R. W. Adey, B. Jessup, and W. Oswald. 1996. A controlled stream mesocosm for the

tertiary treatment of sewage. Ecological Engineering 6 : 149-169.

Crawford, S., A Chiovitti, J. Picket-heaps, and R. Wetherbee. 2009. Micromorphogenesis during

diatom wall formation produces siliceous nanostructures with different properties.

Gates,

J. Phycology 45: 1353-1362.

D. 1980. Biophysical Ecology. Springer-Verlag, Berlin. 611 pp.

Graham,L. and L. Wilcox. 2000. Algae. Prentice-Hall, Upper Saddle River, 640 pp.

Mulbry, W., S. Kondrad, C. Pizarro and E. Kebede-Westhead. 2008. Treatment of dairy manure

Effluent using freshwater algae: algal productivity and recovery of manure nutrients using pilot-scale algal turf scrubbers. Bioresource technology 99: 8137-8142.

Mulbry, W., E. Kebede-Westhead, C. Pizarro and L. Sikora. 2005. Recycling of manure

nutrients: use of algal biomass from dairy manure as a slow release fertilizer.

Bioresource Technology 96: 451-458.

Ramey, D. and Shang-Tian, Yang. 2004. Production of butyric acid and butanol from biomass.

Final report to DOE for contract DE-F-G02-OOER86106.

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Ryan, C. 2009. Cultivating Clean Energy the promise of algae, NRDC and Terrapin Bright

Green, LLC. pp1-81.

Sheehan, J. T. Dunahay, J. Benneman and P. Roessler. 1998. A look back at the U.S. Department

of Energy‟s aquatic species program: biodiesel from algae. Golden, Colorado, National

renewable Energy Institute.

NREL/TP 580 24190.

Stewart, E. 2006. Preliminary engineering assessment for a comprehensive algal turf scrubber-

based nutrient control program for the Suwannee River in Florida. 19pp + 18 sheets.

Van den Hoek, C. 1995. Class Bacillariophyceae. (In) Mann, D. and J. Jahns. Algae, An

Introduction to Phycology. Cambridge University press.

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Appendix to:

ATS to Energy Project

A. Algae of the ATS Test Systems, Smithsonian Institution

B. Susquehanna Project, University of Maryland

C. Ozark Highlands Project, University of Arkansas

D. Biomass Processing, Western Michigan University

E. Project Extension (Chesapeake Algae Project, ChAP/Chesapeake Algae Consortium): organizations listed above, plus Blackrock Energy, StatoilHydro, Exelon Power, Constellation Energy, HydroMentia, Ecological Systems Technology University of William and Mary/VIMS,

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Appendix A The ATS Energy Project

Algae of ATS Test Systems

Haywood Dail Laughinghouse IV

Department of Botany National Museum of Natural History Smithsonian Institution Washington, DC 20560 and Department of Environmental Science and Technology University of Maryland College Park, MD 20742

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Background

The word „algae‟ was first used by Linnaeus in 1753 in Species Plantarum to denote a group of cryptogams (plants with hidden reproduction, without flowers or cones). However, the organisms that are englobed by this term do not constitute a singular evolutionary taxonomic group and are polyphyletic including both prokaryotic and eukaryotic nucleate organisms, within many distant clusters. A common characteristic within all the algae is that they contain the green pigment chlorophyll a within their cells, providing photosynthesis and the ability to maintain an autotrophic life, although some algae can also be heterotrophic during parts of their lives. Some algal groups, including the diatoms that figure prominently on the ATS systems of this project, also have accessory pigments (brown, yellow, blue, and red), capable of capturing solar energy and providing the coloration of the group that is other than green.

Algae are a highly diverse group of organisms, and can be found in an array of different ecosystems. Most of the large macroscopic species live in marine waters (e.g. kelps and rock weeds), while the microscopic species have a wider distribution, though these microscopic, basically single-celled species, can accumulate in colonial and filamentous types and can form macroscopic mats or „algal turfs‟. Many species occur in fresh and brackish waters, though they can also be found in snow, thermal vents, humid soils, desert crusts, in/on plants, animals, rocks, etc. Basically, we live in an algal world.

In the aquatic environment algae are mostly planktonic or benthic. Most benthic (periphytic) algae are either blue-green algae (Cyanobacteria), green algae (Chlorophyta), diatoms (Bacillariophyta), or red algae (Rhodophyta), though on the freshwater ATS systems of this study no rhodophytes were observed, however the red algal genus Polysiphonia was encountered on the estuarine systems, especially that of Great Wicomico River, which has around 14‰ salt.

The colonization by algae of an aquatic surface is a linear process of the settlement rate directed by the size and type of the source system (if this is dominated by bedrock, SAVs,

sediment, etc.), substratum texture, light intensity, and water velocity (see Biggs, 1996). In our cases, the source waters favored by the ATS system, tends to be dominated by diatoms (Class Bacillariophyceae) (see Figures below obtained by microscope counting). These are important

components of many algal communities.

usually occurs where first an organic matrix with bacteria will develop on a substrate, with small

adnate diatoms, then larger colonial diatoms, and so on up to larger filamentous greens (Chlorophytes). Since there is a weekly harvest on ATS floways, we tend to keep the system at the intermittent stage of colonial diatoms. Since the primary objective in ATS systems is to maximize algal growth rate, and therefore nutrient removal, leaving algae on the floway too long will cause sloughing and loss in biomass (Biggs, 1996).

In natural short-term communities, a succession

47

Diatoms are unicellular organisms that can sometimes form colonies which are filamentous, radial, or spiral. Their bivalve cell wall (resembling petri dish), is characterized by having a polysaccharide cell wall containing silica (frustule). Both valves are surrounded by girdle bands, which also contain silica. In certain species, like the tube-forming Berkeleya

rutilans, numerous frustules are found within a thick polysaccharide mucilage/sheath.

containing chlorophylls a and c, diatom plastids have the brown to yellow pigments fucoxanthin, diadinoxanthin, and diatoxanthin, allowing for the golden-brown color of the Bacillariophyceae. The group stores its reserve principally as chrysolaminarin (glucose β 1-3 ramified in β 1-6), but the cells can also accumulate lipids (Reviers, 2006), hence its large use in many studies for biodiesel production. Diatoms are capable of moving (gliding) on substrates by excreting mucilage through their raphes (fissure running the length of the valve of some pennate diatoms). This mucilage is involved in their adhesion to the substrate, as well. Another interesting feature is that some diatom species are capable of burying themselves under the sediment at times, controlled by an internal „biological clock‟ (Hoek, 1995).

Besides

by an internal „biological clock‟ (Hoek, 1995). Besides On the left, Luticola cf. geoppertiana from Muddy
by an internal „biological clock‟ (Hoek, 1995). Besides On the left, Luticola cf. geoppertiana from Muddy

On the left, Luticola cf. geoppertiana from Muddy Run ATS seen through SEM, demonstrating some of the characters used for morphological analysis. On the right, Cymbella tumida with many Navicula seen using LM and phase contrast.

Diatom frustules are extremely ornamented, some visible in the light microscope (LM), like raphe, striae, and nodules, but others only visible under scanning electron micrscopy (SEM), like pores, areolae, channels, and diverse processes like (rimoportulae, fultoportulae, spines) which are found internally or externally in the valves. The presence, location, disposition, and number of these structures are the characters used to identify the genera and species of the Baciallariophyceae (Barber & Haworth, 1994), since these are different in evolutionary lineages.

ATS floways also contain chlorophytes, a.k.a green algae. This group of algae differentiates from the diatoms by containing chlorophylls a and b, as principal photosynthetic pigments, though in some cases the accessory pigment siphonoxanthin or lutein are found (Sze, 1993). Like the higher plants that green algae gave rise to, chlorophytes present a green

48

coloration since, in general, they do not contain other pigments masking the color. This group usually uses starch as a reserve. The morphology of this group is diverse, containing unicellular, colonial, coenobial, flagellated, filamentous, (pseudo)parenchymatous, etc. species. In the algal turfs, besides metaphytic chloroccocalean ceonobial species like Pediastrum, Scenedesmus, and Desmodesmus, true filamentous forms were found such as Ulothrix, Cladophora, Stigeoclonium, Oedogonium, and Rhizoclonium.

Oedogonium is a common green algae of relatively calm freshwater habitats, which was true in the ATS floways of Muddy Run, from the middle to the bottom. The filament (thallus) is unbranched and attached by a holdfast. Besides interesting morphologically characters, like striations or caps in the cells from cell divisions, reticulate chloroplasts, and large vacuoles, among others, this alga has internal thickening of its cell walls following cell divisions. This ring consists of noncellulosic polysaccharides (hemicellulose) (Sze, 1993), and cellulose is deposited on the inside of this ring.

and cellulose is deposited on the inside of this ring. Photomicrograph of „green‟ algal clump from

Photomicrograph of „green‟ algal clump from Muddy Run ATS. a) Cladophora glomerata with epiphytes (diatom Cocconeis), b) Oedogonium sp. whose wall doesn‟t allow the attachment of epiphytes.

Numerous Cladophora spp. were seeded on the ATS floways, though great long term

success was not achieved.

(pers. comm.) comments that for Cladophora to grow well in an environment, both high nutrients and high light intensity, is required while other authors comment that there is a seasonality effect to Cladophora, where late winter/early spring is often diatom dominated, progressing with cyanobacteria (e.g. Phormidium sp.) in the summer and patchy growth of large filamentous algae, like Cladophora in late summer (see Biggs, 1996). A seasonal cycle resembling this was seen on the Muddy Run ATS floways and the Springdale floway increased to 35% of their relative biomass in green algae towards the end of the summer season. Each cell

This alga is branched and also attaches by a holdfast. B. Whitton

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of Cladophora is surrounded by a thick cellulosic cell wall which is continuous throughout the filament; it lacks a mucilaginous sheath, allowing for the growth of epiphytes. This is different than Oedogonium or Spirogyra that contain a sheath that does not permit epiphytic growth.

Cyanobacteria were also found on the floways, though usually presenting less than 5% of total biomass. The most common species were of the Phormidium complex. This group, by its name, is a prokaryotic alga, possessing chlorophyll a and photosystems I and II, carrying out photosynthesis in the presence of oxygen. They possess phycobilisomes with accessory pigments such as phycoerythrin, phycocyanin, and allophycocyanin (Reviers, 2006). These produce a typical blue-green color for the group. Morphologically, the group is simple, difficulting precise identification of many species, although the vegatitive structure can be coccoid, colonial or filamentous, and contain mucilaginous sheaths. There are various types of storage in the cyanobacteria: nitrogen reserves are constituted of cyanophycin and carbon reserves formed by cyanophycean starch and lipids, you can also find polyphosphate granules in the cells. Their cell walls are composed of peptidoglycan, characteristic of eubacteria. Of ecological importance is the capability of cyanobacteria to metabolize atmospheric nitrogen, particularly when nitrogen concentrations in the water column are very low; this is facilitated by the presence of heterocytes, although species without this cell are also capable of using atmospheric nitrogen.

this cell are also capable of using atmospheric nitrogen. Phormidium sp. from periphyton collected at Muddy

Phormidium sp. from periphyton collected at Muddy Run ATS

Sampling

Throughout the study period, samples of periphyton were collected from all of the ATS Systems of this project and subsequently analyzed at the National Museum of Natural History (NMNH). For qualitative analyses, periphyton was scraped off the screen with a spoon at various parts of the floway to have an understanding of the full complexity of the algal community. For quantitative analyses, top, middle, and bottom portions of the system of a

50

known size (between 50-100 cm 2 ) were scraped and sent to NMNH for analysis or portions of the screen were sent and then processed in the same manner.

Quantitative analysis

Samples of thick algal mats were homogenized (blended) in distilled water, where the final volume was recorded. A subsample of 100ml was taken and standard lugol‟s solution was added to sediment and stabilize the samples for analysis preparing it for counts in the inverted microscope. After about 3 hours, or enough time for lugol to penetrate the algal cells in the subsample, the sample was homogenized by shaking slowly back-and forth for 1 minute, and 5ml was placed in a sedimentation chamber of the same volume. This was let sit for a minimum of 6 hours, so that the algal cells could sediment to the bottom of the chamber, allowing visualization in the Zeiss inverted microscope for counting.

Algal counts of periphyton were conducted on a Zeiss inverted microscope, counting a minimum of 500 cells/valves. These were recorded and a formula proposed by Wetzel & Likens (1979) modified by Schwarzbold (1992) was used to determine the number of algal cells p/cm 2 :

N = n . V / v . 1/S

where:

N

= number of individuals per cm 2

n

= total number of individuals counted in the sample

V

= volume of the sample with scraped material in ml

v

= volume of the counted fields

S

= surface of the substrate in cm 2

Qualitative analysis

Diatoms were cleaned in preparation from LM and SEM, this is a different procedure than just visualization that is conducted for soft algae (green and blue-green algae). Twenty ml of sample were removed and boiled in hydrogen peroxide 30% in a l:1 sample ratio, adding potassium dichromate during the process to speed oxidization, until returning to the original volume of 20 ml. The cleaned material was then concentrated in a centrifuge, and rinsed several times until reaching a neutral pH (c. 10x). This material was diluted into 3 different dilutions, air dried on coverslips, and processed for either LM or SEM analysis.

For LM, the coverslips were let dry overnight, and then mounted onto glass slides with the high infraction index (1.704) mounting medium Cargille Meltmount ™. The samples were then analyzed under 400x or 1000x (oil immersion) using an Olympus Bx50 compound microscope with phase contrast. Morphological characteristics were recorded for the taxa for specific delimitation, i.e. valve/cell diameter and length, diameter of central area, striae length, density, and number.

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For SEM, coverslips were mounted on specimen stubs and sputter-coated with a gold/palladium alloy (10nm) and analyzed using a Leica Stereoscan 440 SEM.

The algae were qualitatively analyzed according to specific literature for each group:

Bacillariophyta (Krammer & Lange-Bertalot, 1986, 1988, 1991, 1991; Cox, 1996; Round et al., 1990); Cyanobacteria (Komarek & Anagnostidis, 1998, 2005); Chlorophyta (Prescott, 1982; Bicudo & Menezes, 2005; John & Williamson, 2009) in addition to using some regional floras (Prescott, 1982). Algal counts were conducted on the most dominant algae found on each system (cell p/cm 2 ), using the inverted microscope method with sedimentation chambers (Utermöhl, 1931; Lund et al, 1958) and a formula by Wetzel & Likens (1979) modified by Schwarzbold (1992).

Results

Overall, diatoms dominated the floways in all of the ATS systems studied, in both biomass and species richness, oscillating from a little over 60% to over 95% of the total biomass during harvest periods, depending on the system and/or the season. The first two charts on the following page demonstrate the percent biomass of the different algal groups found on the ATS systems in Springdale, AR and Gloucester Point, VA, followed by a diagram with species diversity in April, 2009 of both the 1% slope wood and 2% slope aluminum floways at the Muddy Run, PA site.

Some of the most abundant and frequent species of diatoms found in the freshwater ATS systems are Melosira varians, Diatoma vulgare, Fragilaria spp. (ex. F. capucina), Gomphonema spp. (ex. G. olivaceum and G. truncatum), Navicula spp. (ex. N. gregaria, N. lanceolata), and Nitzschia spp. (ex. N. cf. dissipata). In the estuarine systems, the diatoms encountered are the tube-forming Berkeleya rutilans (and some Navicula sp. associated), Melosira nummoloides, Licmorpha spp., Gyrosigma sp., and Nitzschia sp. Many of these common diatom elements are shown in the following figures.

As commented previously, diatoms, like other algae, can be found naturally in many environments, though Gyrosigma sp., Navicula spp., and Nitzschia spp. tend to be common on the sediment or strictly part of the silt flora, also on rocks which are covered with fine sediment. Diatoma vulgare and Gomphonema spp. are commonly found growing on rocks (sometimes plants) in rivers, and are capable of massive growths. Fragilaria spp. are found in various habitats, like the plankton in lakes or on sediments and plants in rivers. Melosira varians forms „loose flocs‟ on river banks especially in the slower moving parts of the river. These are easily broken apart, releasing the frustules into the water column of the river, where they are able to move downstream. M. varians is a freshwater species, however in brackish environments,

52

species such as M. nummoloides can be found in similar habitats (Kelly, 2000). Berkeleya rutilans grows in estuarine-marine environments, attached to the rocks, forming colonies resembling macroscopic seaweeds, though having a free living state during times of the year (Lobban, 1984).

References

Barber, HG & Haworth, EY (1994) A guide to the morphology of the diatom frustules. Freshwater Biological Association Scientific Publication 44: 1-112.

Bicudo, CEdeM & Menezes, M (org.) (2005) Gêneros de Algas de Águas Continentais do Brasil: Chave para Identificação e Descrições. São Carlos: RiMa, 489pp.

Biggs, BJF (1996) Patterns in Benthic Algae of Streams. In: Stevenson, RJ; Bothwell, ML; Lowe, RL eds., Algal Ecology: Freshwater Benthic Ecosystems. San Diego: Academic Press, p. 31-56.

Cox, EJ (1996) Identification of Freshwater Diatoms from Live Material. London: Chapman & Hall, 158 pp.

Hoek, C van den; Mann, DG; Jahns, HM (1995) Algae: An introduction to phycology. Cambridge: Cambridge University, 627pp.

John, DM & Williamson, DB (2009) A Practical Guide to the Desmids of the West of Ireland. Galway, Ireland: Martin Ryan Institute, 196pp.

Kelly, M (2000) Identification of common benthic diatoms in rivers. Field Studies 9: 583-700.

Komárek, J & Anagnostidis K. (1998) Cyanoprokaryota 1.Teil: Chroococcales. In: Ettl H., Gärtner, G; Heynig, H; Mollenhauer, D eds, Süßwasserflora von Mitteleuropa 19/1, Jena:

Gustav Fischer, 548 pp.

Komárek, J & Anagnostidis K. (2005) Cyanoprokaryota 2.Teil: Oscillatorialles. In: Budel, B; Krienitz, L; Gärtner, G; Schagerl, M. eds., Süßwasserflora von Mitteleuropa 19/2, München:

Elsevier GmbH, 758 pp.

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Krammer, K & Lange-Bertalot, H (1986) Bacillariophyceae 1. Teil: Naviculaceae. In: Ettl H., Gerloff, J; Heynig, H; Mollenhauer, D eds, Süßwasserflora von Mitteleuropa 2/1, Stuttgart:

Gustav Fischer Verlag, 876 pp.

Krammer, K & Lange-Bertalot, H (1988) Bacillariophyceae 2. Teil: Bacillariaceae, Epithemiaceae, Surirelliaceae. In: Ettl H., Gerloff, J; Heynig, H; Mollenhauer, D eds, Süßwasserflora von Mitteleuropa 2/2, Stuttgart: Gustav Fischer Verlag, 596 pp.

Krammer, K & Lange-Bertalot, H (1991) Bacillariophyceae 3. Teil: Centrales, Fragilariaceae, Eunotiaceae. In: Ettl H., Gerloff, J; Heynig, H; Mollenhauer, D eds, Süßwasserflora von Mitteleuropa 2/3, Stuttgart/Jena: Gustav Fischer Verlag, 576 pp.

Krammer, K & Lange-Bertalot, H (1991) Bacillariophyceae 4. Teil: Achnanthaceae, Kritische Ergänzungen zu Navicula (Lineolatae) und Gomphonema Gesamtliteraturverzeichnis Teil 1-

4. In: Ettl H., Gartner, G; Gerloff, J; Heynig, H; Mollenhauer, D eds, Sußwasserflora von

Mitteleuropa 2/4, Stuttgart/Jena: Gustav Fischer Verlag, 437pp.

Lobban, CS (1984) Marine tube-dwelling diatoms of eastern Canada: descriptions, checklist, and illustrated key. Can. J. Bot. 62: 778-794.

Lund, JWG; Kipling, C; Le Cren, ED (1958) The Inverted Microscope Method of Estimating

Algal Numbers and the Statistical Basis of Estimations by Counting. Hydrobiologia 11: 143-

170.

Prescott, GW (1982) Algae of the Western Great Lakes Area. Revised edn. Dubuque, Iowa:

Brown, WMC, 977pp.

Reviers, B de (2006) Biologia e filogenia das algas. Trans. Iara M. Franceschini. Porto Alegre:

Artmed Editora, 280 pp.

Round, FE; Crawford, RM; Mann, DG (1990) The Diatoms: Biology and Morphology of the Genera. Cambridge: Cambridge University Press, 747pp.

Schwarzbold, A. (1992) Efeitos do regime de inundação do rio Mogi-Guaçu (SP) sobre a estrutura, diversidade, produção e estoques do perifíton de Eichornia azurea (Sw) Kunth da Lagoa do Infernão. PhD dissertation, Universidade Federal de São Carlos, São Carlos,

237pp.

Sze, P (1993) A Biology of the Algae. 2 nd ed. Dubuque, Iowa: WC Brown Publishers, 259pp.

Utermöhl, H (1931) Neue Wege in der quantitativen Erfassung des Planktons. Verh. Internat. Verein. Limnol. 5: 567-596.

Wetzel, RG & Likens GE (1979) Limnological Analyses. Philadelphia: Saunders, 357pp.

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Relative biomass of the different algal groups of the ATS Springdale floway during the indicated

Relative biomass of the different algal groups of the ATS Springdale floway during the indicated period. Most common species of each group are listed with percentages.

common species of each group are listed with percentages. Relative biomass of the different algal groups

Relative biomass of the different algal groups of the ATS Springdale floway during the indicated period. Most common species of each group are listed with percentages.

55

Number of Species

CYANOBACTERIA

BACILLARIOPHYTA

ZYGNEMAPHYCEAE

CHLAMYDOPHYCEAE

CHLOROPHYCEAE

RAPHIDOPHYCEAE

CRYSOPHYCEAE

EUGLENOPHYCEAE

Wood and Aluminium Floways species group comparison

70

60

50

40

30

20

10

0

Floways species group comparison 70 60 50 40 30 20 10 0 Wood Aluminium Total Species
Floways species group comparison 70 60 50 40 30 20 10 0 Wood Aluminium Total Species
Floways species group comparison 70 60 50 40 30 20 10 0 Wood Aluminium Total Species
Floways species group comparison 70 60 50 40 30 20 10 0 Wood Aluminium Total Species
Wood Aluminium Total
Wood
Aluminium
Total

Species richness comparison between the two ATS floways at Muddy Run.

Great Wicomico River ATS - Virginia 3-D screen 11/4/2009 Chlorophyt a 7% Cyanobacte ria 10%
Great Wicomico River ATS - Virginia
3-D screen
11/4/2009
Chlorophyt
a
7%
Cyanobacte
ria
10%
Bacillarioph
yta
83%

Great Wicomico River ATS - Virginia

2-D screen 11/4/2009 Chlorophyta 18% Bacillarioph yta 82%
2-D screen
11/4/2009
Chlorophyta
18%
Bacillarioph
yta
82%

Relative biomass counts conducted on samples from the Great Wicomico River comparing the 2-D and 3-D screens in use. Besides difference in diatom species composition in each sample, there was a difference of taxonomic groups. More filamentous and erect diatom species found on the 3-D, compared to more adnate and erect species on the 2D. More samples need to be analyzed to have a better understanding of the significance of this difference.

56

1
1
3
3
5
5
2
2
4
4
6
6

1 Berkeleya rutilans; 2 Melosira nummoloides; 3 Nitzschia sp.; 4 Melosira spp.; 5 Cocconeis sp.

on Ceramium sp.; 6 Licmorpha sp.

57

1
1
3
3
5
5
2
2
4
4
6
6

1 Surirella sp.; 2 Encyonema sp.; 3 Diatoma vulgare; 4 Achnanthes sp.; 5 Eunotia sp. 6 Nitzschia sp.

58

1
1
3
3
5
5
2
2
4
4
6
6

1-2 Biofilm of diatoms; 3 Cymbella sp

6 Pediastrum boryanum

;

4 Gomphonema truncatum; 5 Melosira varians;

59

1
1
4
4
7
7
2
2
5
5
b a 8
b
a
8
a b 3
a
b
3
6
6
9
9

1 Cladophora glomerata; 2 Ulothrix sp.; 3a C. glomerata; 3b Oedogonium sp.; 4 Klebsormidium sp.;

5 Melosira varians; 6 Uronema sp. 7 Phormidium sp.; 8a Spirogyra sp.; 8b Rhizoclonium sp.; 9 Diatoma vulgare

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Appendix B

Final Report on the Susquehanna River Algal Turf Scrubber Project

December 2009

Patrick Kangas (1), Walter Mulbry (2), Philip Klavon (1), Dail Laughinghouse (1)

1) Environmental Science and Technology Department

University of Maryland

College Park, Maryland

2) United States Department of Agriculture

Beltsville Agricultural Research CenterBeltsville, Maryland

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Main Findings of the Report:

Over a growing season of 8 months, net biomass production averaged 14.0 grams dry weight/m2/day for the aluminum ATS and 11.7 grams dry weight/m2/day for the wooden ATS with a peak of 24.5 grams dry weight/m2/day on 7/28/08.

These net biomass production values are higher than any published values from natural aquatic plant communities in the Chesapeake Bay.

Biomass production was composed of the vacuum harvest (52% of the total production for the aluminum ATS and 29% for the wooden ATS), the “greenwater” (42% of the total production for the aluminum ATS and 66% for the wooden ATS) and the slough (6% of the total production for the aluminum ATS and 5% for the wooden ATS).

During 2009 at peak metabolism the wooden ATS decreased nutrient concentrations to a greater degree (nitrate-N 27%, orthophosphate-P 50%, total N 37% and total P 17%) than the aluminum ATS (nitrate-N 18%, orthophosphate-P 17%, total N 28% and total P 9%).

Algal biomass averaged between 2-3% nitrogen content and between 0.25-0.35% phosphorus content, depending on the component (vacuum harvest, greenwater, slough).

Net production based on ecosystem metabolism measurements of the two ATS varied seasonally from less than 1 to 8.6 grams oxygen/m2/day.

Species diversity of the ATS was high: 195 species from six algal phyla on the aluminum ATS and 196 species from five algal phyla on the wooden ATS

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NARRATIVE

This is the final report on the Susquehanna River ATS (algal turf scrubber) project. The proposal for the project was submitted in late spring of 2007 and the intention was to have the ATS operational by early spring 2008. The summer of 2007 was spent searching for a suitable site with close proximity to the river, no shading and security. Contact was eventually made with the Exelon Energy Corporation, which operates several power plants on the lower Susquehanna River. The manager of the Conowingo hydroelectric plant became interested in the project and she assisted in establishing and conducting the project since the first contact. Meetings were held with Exelon administrators during the fall and winter in order to secure corporate permission to operate the ATS on Exelon property. After extensive communications between the Exelon Corporation, the Smithsonian Institute’s National Museum of Natural History and the University of Maryland, a suitable agreement was established for the research to be carried out at the Muddy Run hydroelectric power plant in Lancaster County, Pennsylvania. The Hydromentia Corporation of Ocala, Florida fabricated the ATS from aluminum and had it shipped to the site in late spring of 2008. The system was constructed at Muddy Run with assistance from Exelon staff and from the company Living Ecosystems of Easton, Maryland. The dimensions of the original system were 1’ by 300’ and it was positioned at a 2% slope.

Construction of the ATS was completed on 6/6/08 with water input from the Muddy Run Reservoir. Rocks from the Susquehanna River at Muddy Run were added to the ATS to both provide a seed source for benthic algae and to submerge the ATS screen, which was not physically attached to the trough or bed of the ATS. Wire braces were also installed in order to keep the screen submerged. Benthic algae from a local creek, which were placed in bags fashioned from the ATS screen material, was also used to seed the system. Local streams were searched for Cladophora, which was not found at the Muddy Run site. Two nearby streams with Cladophora were located and rocks from these streams were added to the system with the intention of adding this genus to the scrubber community.

An algal community developed relatively rapidly and the system was first harvested on 6/23/08 to initiate the study of biomass production and nutrient dynamics. Measurements of biomass production, or net primary production, began on 6/28/08 and continued throughout the growing season approximately every five-seven days.

An additional ATS was constructed out of wood with fiberglass coating by Living Ecosystems with the same dimensions as the original aluminum ATS. This system was installed parallel to the aluminum ATS at a 1% slope in order to investigate the effect of ATS slope on productivity and nutrient dynamics. The wooden ATS also differed from the aluminum ATS in mesh size of the screen used to grow algae: the wooden ATS had a mesh size of 0.04 cm2 while the original aluminum ATS had a mesh size of 0.25 cm2. The different mesh sizes were used to investigate the effort of screen surface area (which was assumed to be inversely related to mesh size) on productivity. The wooden ATS was constructed in the early fall 2008 and it was first harvested on 10/16/08. Both ATS were operated through the fall and the last harvests were taken on 12/8/08.

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The intention was to operate both ATS through the winter but cold temperatures (see Figure 1) caused the water in the systems to freeze periodically. Freezing started at the bottom of the ATS and this caused water to spill on to the ground creating a sheet of ice around the systems. The ice was a safety hazard so the ATS were shut down several times during the winter and early spring. Thus, no harvests were taken during the winter because of the freezing problem.

Water flow was restored in March 2009 and algal growth occurred immediately. The systems were first harvested on 4/14/09. After an annual cycle of data was gathered from the original aluminum ATS at the end of July 2009, its slope was dropped to 0.5% in order to investigate the effect on productivity through the late summer and fall. The last harvest of the study occurred on 10/29/09 once an annual cycle of data had been gathered on the wooden ATS. After this time, emphasis in the project shifted from field work to analysis of data and to final report preparation.

A four month-long infestation of herbivorous chironomid fly larvae occurred in 2009 that dominated the ecology of the systems. This infestation was somewhat surprising since no chironomids were observed in the systems in 2008. The chironomids first appeared in late May 2009 in the aluminum ATS and they cleared algae from the screens throughout the summer. The larvae were present in the wooden ATS during this time but they did not cause visible eat-outs until late July. The intensity of the herbivory was not uniform throughout the ATS but the chironmids did consume enough algae to cause large areas of bare screen in the systems. Herbivory seemed to be most evident in the lower and middle sections of the ATS. Several control techniques were used to manage the infestation including altered harvest frequencies, short term (usually 3 day duration) drainage of the systems and use of a bacterial insecticide. These control techniques did reduce chironomid densities but the fly larvae always returned. The chironomids seemed to enter the system through the input water, though it is possible that egg laying by adults within the raceways could have contributed to the population. The infestation disappeared in mid-September, presumably due to seasonal completion of the fly life cycle. After this time algal growth increased and full coverage of the screens developed.

METHODS

Basic Water Quality

Water quality parameters were measured before each harvest in the late morning or afternoon in order to quantify the maximum effect of metabolism of the algal community. Measurements were made at the top of the system, where water enters from the Muddy Run reservoir, and at the bottom of the system, as the water flows into the river. The difference between bottom and top values indicates the effect of algal metabolism as water passes over the turf. Water temperature, dissolved oxygen concentration and percent saturation of dissolved oxygen in the water were measured with a YSI meter and pH was measured with an Accumet meter. On 10 dates, water quality parameters were measured over a diurnal cycle in order to assess ecosystem metabolism of the turf communities.

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Biomass Harvest

Algal biomass was harvested in sample sections along the length of the ATS in order to identify possible longitudinal changes in the community. Several different patterns of harvests were tried initially. The final pattern of harvest was established on 7/28/08 and it was used throughout the study. For the aluminum ATS biomass was harvested in two adjacent 10’ sections of the scrubber near the top of the system (T1 at 50 to 60 feet downstream from the surge box, T2 at 60 to 70 feet), in the middle (M1 at 170 to 180 feet, M2 at 180 to 190) and at the bottom of the system (B1 at 280 to 290 feet and B2 at 290 to 300 feet). This pattern of harvest included replication of sampling (n=2) at three different locations along the longitudinal gradient of water input at the top of the system to the water output at the bottom of the system. Each individual section of the aluminum ATS had an area of approximately 0.9 m2 (the one foot wide trough x a 10’ section between supports on the trough) while each section of the wooden ATS had an area of approximately 0.7 m2. For the aluminum ATS the total sample area at each harvest was then 5.4 m2 which is 20 % of the total scrubber area (27.6 m2) for the aluminum ATS. At the time of harvest the water input to the ATS was turned off at the inflow faucet and the scrubber was allowed to drain for between ½ - 1 hour. Harvesting was done with a wet/dry vacuum. Each sample section of the trough was vacuumed, starting at the top of the system and working sequentially downstream. The entire ATS area (except for the first section below the surge box which was maintained as an unharvested refuge) was harvested but only the biomass production samples were retained for measurements. After a sample section was vacuumed, the resulting slurry was dewatered by sieving the harvested material through a 3mm mesh nylon netting (Aquatic Ecosystems, Apopka, FL). The biomass retained in the net was air-dried at 25 degrees C using an electric fan, oven-dried at 70 degrees C for 24 hours and then weighed. Data for biomass production or net primary production were calculated by dividing the oven-dried mass by the number of days between harvest dates. Throughout the study 235 vacuum harvest samples were analyzed for the aluminum ATS and 160 were analyzed for the wooden ATS.

Two other components of the biomass production of the algal turf community were assessed: algal biomass suspended in the water that was sieved through the nylon netting (termed “green water”) and algal biomass that broke off from the turf between harvests (termed “slough”). For biomass in green water, the volume of sieved water was measured at the time of harvest and a one liter sample was collected and returned to the laboratory. The biomass was allowed to settle out of suspension in the sample bottle creating a dense layer in the bottom of the bottle. The overlying water was removed by vacuuming or decanting and the remaining slurry that contained the biomass was spread out on a tray lined with a plastic film in which the water was allowed to evaporate. The biomass remaining after evaporation was oven-dried at 70 degrees C and weighed. Biomass production of the green water component was calculated by dividing the biomass by the area of turf that was harvested and sieved and by the number of days between harvest dates. Routinely, green water was combined for paired samples at the top, middle and bottom of the ATS resulting in three samples for each harvest date after 7/28/08. Throughout the study 105 greenwater samples were analyzed for the aluminum ATS and 68 were analyzed for the wooden ATS. Biomass that sloughed from the turf between harvest dates was

65

collected in a nylon mesh bag that was attached to the outlet drain pipe at the bottom of the ATS. Material collected in the bag was removed at the time of harvest and it was processed by the same procedure as the biomass harvest collected with the wet/dry vacuum. To calculate the slough production, the biomass collected between harvest dates was divided by the total area of the scrubber and by the number of days between harvests. Throughout the study 23 slough samples were analyzed for the aluminum ATS and 10 were analyzed for the wooden ATS.

Ecosystem Metabolism

Diurnal curve analysis of dissolved oxygen concentration was used to estimate ecosystem metabolism and to help assess the functioning of the ATS systems. Nine diurnal curves were analyzed during the study period covering all of the seasons of the year. The basic water quality parameters of temperature, dissolved oxygen concentration, percent saturation of oxygen and pH were measured at various time intervals over a 24-hour time period to create the diurnal curves. Ecosystem metabolism was estimated using the dissolved oxygen concentration data. The concentration at the top of the system (input water) was subtracted from the concentration at the bottom of the system (output water) and this difference was divided by the turnover time of water in the system (an average of 10 minutes was used) to calculate rates of oxygen change. These rates of change in oxygen concentration were then plotted over 24 hours and graphically integrated to estimate metabolism. This approach is essentially a modified version of the standard upstream-downstream technique for measuring ecosystem metabolism in flowing water systems (Odum 1956).

No corrections to the rate-of-change curves were made for ambient diffusion based on three reasons. First, percent saturation of dissolved oxygen was usually near 100% when top (input) and bottom (output) samples were averaged. Second, it was assumed that the fast turnover time of water in the system (about 6-20 minutes) precludes any major changes in oxygen concentration due to diffusion. Third, upstream-downstream sampling of dead turfs, either during the winter or after drainage for chironomid control in the summer of 2009, indicated little evidence of physical diffusion.

Nutrients

Water samples were collected at the inflow and at the outflow of the ATS before harvests. These samples were acidified and stored, usually at 4 degrees C, prior to analysis for total nitrogen (TN), nitrate-nitrogen (NO3-N), total phosphorous (TP) and orthophosphate-phosphorous (PO4-P). The water samples were also analyzed ammonium-nitrogen but the resulting values were below detection limits or non-interpretable. Biomass samples were analyzed for TN and TP after oven-drying.

Algal Community Structure

Samples of algae from the scrubber were collected periodically and examined with a compound microscope for the purpose of describing the structure of the community at the time of almost every harvest. Two or three samples from each turf along with a sample of the green water and a sample of the slough were routinely collected during each harvest and these were examined to rapidly assess the

66

dominant alga taxa and the overall condition of the system. Samples were collected for a more in-depth assessment of the species composition of the turfs on 7/23/08, 8/1/08, 10/27/08, 4/7/09, 6/12/09, 7/17/09, 10/9/09. These samples were carefully examined in order to construct a species list of algal taxa found on the scrubber.

RESULTS

Water Quality and Quantity

Water input to both of the ATS came from the Muddy Run Reservoir, which is filled from and drained to the Susquehanna River on a daily basis in the operation of Exelon’s pumped-storage hydroelectric facility. The quality of the input water to the ATS was thus modified from the ambient river conditions by a relatively short retention time in the reservoir. At one seasonal extreme, during the summer, input dissolved oxygen concentration was low at around 4 mg/l with about 50% saturation and pH was about

7.5. At the other seasonal extreme, during the winter, input dissolved oxygen concentration was high at

around 12 mg/l with about 90% saturation and pH was about 8.0. Passage of the input water through the ATS increases dissolved oxygen concentration, percent oxygen saturation and pH in proportion to ecosystem metabolism of the ATS, as will be discussed in a later section of this report. During mid-day these increases are dramatic with dissolved oxygen concentration and percent saturation more than doubling and pH increasing by nearly two units from top to bottom of the ATS raceways. These increases are especially noteworthy given that the turnover time of water in the systems was between 7 and 10 minutes.

Average nutrient concentrations (n=17) of the input water were as follows: 0.89 mg/l NO3-N, 1.38 mg/l TN, 0.06 PO4-P, and 0.12 mg/l TP. These nutrients were reduced by uptake in algal growth as the water passed along the ATS raceway. Mid-day removal percentages are given in Table 1. These data are point measurements within the diurnal cycle and they represent the maximal uptake rates since uptake is proportional to ecosystem metabolism and ecosystem metabolism is highest at mid-day.

Data on water flow rates and turnover times for the raceways are given in Table 2. The flow rate was about 1 liter/second throughout the study but it was reduced slightly after the wooden ATS was constructed in late 2008. The water turnover rates were relatively fast at between 6 and 20 minutes. This represents the time it takes water to move from the top to the bottom of the raceway on average. Turnover time was strongly influenced by water depth, which was inversely related to the slope of the raceway.

Biomass Production

There were three components of algal biomass production for the ATS based on the methods described above: the biomass harvested with the wet/dry vacuum (vacuum harvest), the biomass contained in the water drained from the vacuum harvest (green water) and the biomass that sloughed off the turf between harvests and that was collected in the net at the bottom of the system (slough). Overall biomass production data are summarized in Tables 3a and 3b with seasonal and growing season

67

averages. Average total daily production was slightly higher for the aluminum ATS (14.0 g/m2/day) in comparison with the wooden ATS (11.7 g/m2/day). Peak production occurred in the summer months for both ATS. The highest daily production occurred on the aluminum ATS on July 28, 2008 at 24.5

g/m2/day.

Vacuum harvest contributed about half of the growing season production for the aluminum ATS and about 30% of the total for the wooden ATS. These samples were dominated by a mixture of filamentous green algae (Spirogyra was dominant) and filamentous diatoms (Melosira was dominant) along with pennate diatoms and bluegreen algae (Oscillatoria and Phormidium).

Green water biomass production contributed 42% of the aluminum ATS production and 66% of the wooden ATS production. These samples were strongly dominated by fragments of filamentous diatoms and by pennate diatoms, with practically no filamentous green algae. Averaged over the entire study, average greenwater concentration was 14.2 g dry weight/liter and average greenwater volume was 7.4 liters/m2 of turf for the aluminum ATS. Average greenwater concentration was 11.3 g dry weight/liter and average greenwater volume was 8.0 liters/m2 of turf for the wooden ATS.

Slough biomass production contributed about 5% of the total production for both ATS. These samples were strongly dominated by the filamentous green alga, Spirogyra, though all species from the turf were present in small amounts. This portion of the overall production was the most difficult to measure and more study is required for adequate quantification. Problems occurred when the slough bag would become detached between harvests. Also, in the fall season deciduous leaves from nearby trees fell and collected in the slough bags and it was not possible to physically separate leaves from the algae in order to quantify slough.

Nutrient Uptake Rates

Nutrient uptake by the ATS was found by multiplying biomass production rate (g dry weight/m2/day) by the nutrient contents of the biomass (%). These data are illustrated for the aluminum ATS in Figures 2 and 3. Data for biomass production in these figures is from Table 3a. Although the magnitudes of nutrient contents differed between the different components of biomass production, the ratios between nitrogen (N) and phosphorous (P) are about 8 to 1 for the different components. For the aluminum ATS total uptake rates are 0.34 g N/m2/day and 0.04 g P/m2/day.

Ecosystem Metabolism

Diurnal curve analysis of ecosystem metabolism provides a holistic view of the functioning of the ATS. Dissolved oxygen concentration at the bottom of the systems rises during the day due to photosynthesis and falls at night due to respiration. pH at the bottom of the systems also has this diurnal pattern of change but this reflects dissolved CO2 dynamics since pH is inversely proportional, in a non-linear fashion, to CO2 concentration dissolved in the water.

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Values for net production from the diurnal curves are given in Table 4. Values range from about 1 gram of O2/m2/day during the winter months to nearly 10 grams of O2/m2/day in the summer months. An interesting feature of most of the diurnal curves is the minor role played by community respiration in the ATS. Respiration occurs when oxygen change is negative. Day-time respiration is masked by net production, but at night the rate-of-change of oxygen should become negative. Although some negative rates-of-change did occur on some of the diurnal curves, these rates were always relatively low. In part, the negative rates-of-change at night were mitigated by apparent turbulent diffusion at the top of the system due to the wave generation mechanism. This effect was measured to be an immediate increase of 1 2 mg/l of oxygen once the input water enters the system. Corrections for this increase were made in the analysis of the curves. Thus, total oxygen increase at the bottom of the system is partly due to ecosystem metabolism and partly due to physical, turbulent diffusion.

Algal Community Structure

A total of 195 species were identified from six algal phyla on the aluminum ATS over the study period (Figure 4) and a total of 196 species were identified from five algal phyla on the wooden ATS (Figure 5). Many of the species were rare in the context of frequency of occurrence: 103 species or 53% of the total community were found only once (a frequency of 1/26 or 4%) on the aluminum ATS and 92 species or 47% of the total community were found only once (a frequency of 1/22 or 5%) on the wooden ATS. Thus, as is typical of any ecological community, most of the species in the algal turf community were rare.

Five species were found in at least half of the samples on the aluminum ATS (from the genera Navicula, Cyclotella, Spirogyra, Ulnaria and Melosira) and nine species were found in at least half of the samples on the wooden ATS (from the genera Navicula, Diatoma, Ulnaria, Nitzschia, Cyclotella, Melosira, Spirogyra, Syndra and Monorapidium).

DISCUSSION

Overall Productivity

The two ATS raceways at Muddy Run had net biomass productivities that were comparable to other published outdoor studies from the Chesapeake (Mulbry et al. 2008, in press). Growing season averages were also higher than any reported net productivity from a natural aquatic plant community from the Chesapeake Bay (Table 5). The amplified productivities of the ATS over the natural communities is presumably due to the energy subsidies of the engineered design that the ATS receive from the artificial substrate and pulsing water flows but high species diversity may also play a role as will be discussed in the next section.

However, there are reasons to question whether the ATS productivities measured at Muddy Run might be less than what could have been achieved. General predictions for ATS productivity suggested that >30 grams dry wt./m2/day might be possible (Adey and Loveland 2007). In fact, higher productivities have been measured in similar ATS raceways in the more southerly portions of the bay on

69

the Wicomico River in Virginia (W. Adey, personal communication) and at the Virginia Institute of Marine Science at the mouth of the York River (E. Duffy, personal communication).

One possible cause of reduced productivity in the Muddy Run ATS studied here was artificial shading from the walls of the raceways. A preliminary study was made in the late fall 2008 to test this hypothesis. Shading by the walls may be suspected to be significant at this season, due to the reduced angle of the sun in the sky. Algal biomass was harvested from five sections of the aluminum ATS and then these sections were allowed to grow back. At the next harvest biomass in the shaded half of the raceway was gathered separately from biomass in the unshaded half of the raceway. After processing, the results indicated 20% greater production in the lighted half of the system compare to the shaded half. Thus, shading may well be limiting productivity of the ATS, at least during certain times of the year.

One remarkable finding of this study was the relative lack of effect of chironomid herbivory on overall productivity of the ATS. This observation comes from comparing productivity during the summer of 2008, when no chironomids were found on the ATS, to productivity during the summer of 2009, when chironomids were abundant and their feeding created bare areas with no filamentous algae on the screens. Apparently, productivity shifted from filamentous species to single-celled species during the chironomid outbreak and this pattern is indicated to some extent by the changes in proportion of the vacuum harvest (which was dominated by filamentous species) in relation to the greenwater harvest (which may have had greater contribution from single-celled pennate diatoms). This pattern of biomass harvest productivity and herbivory was also found with oxygen-based productivity estimates. In general, there seemed to be some indication that the greenwater proportion of total productivity increases with stress, as noted above with chironomid herbivory, since this proportion was higher in the spring and fall when temperature and/or light levels may be limiting the metabolism of the system. The proportions of total productivity from vacuum harvest, greenwater and slough will have potentially significant implications for commercial-scale harvesting techniques, so this topic deserves further study.

Another hypothesis about limitations on Muddy Run ATS productivities concerned the slough component of biomass harvest. One idea was that slough biomass was being lost in the outlet water and that there was biomass production missing from the totals reported in Table 3. Slough was difficult to measure due to problems with the bags attached to the outlet pipe at the bottom of the raceways. To test the hypothesis of missing slough biomass production, short-term (5 minutes) collections of slough were made over four diurnal cycles in summer-fall 2009. These data averaged 1.4 grams dry wt./m2/day for the aluminum ATS and 1.0 grams dry wt./m2/day for the wooden ATS. These values are consistent with the longer term (5-7 days) collections of slough reported in Tables 3a and 3b so the hypothesis is not supported. The only other possibility is that the missing slough passed through the collection bags, both during the short-term and the long-term collections. If this situation is occurring, finer mesh collection bags will be required to sample this missing biomass and alternative processing techniques will be required to collect, dry and weigh it.

70

Algal Diversity

The sheer magnitude of the diversity of algal species found on the two ATS at Muddy Run is amazing. Nearly 300 species were found on the two scrubbers, which together make up only about 60 m2 of surface area. To some extent the high diversity of algal species on the ATS is due to the species diversity in the source communities for the systems. Ultimately, the source of species is the Susquehanna River, which is a lotic or flowing water ecosystem. However, the river water is passed through the Muddy Run Reservoir before it reaches the ATS. The reservoir is a lentic or standing water ecosystem. Thus, since different species are adapted to lotic vs. lentic ecosystems, these two sources probably contribute to the high diversity of the scrubbers.

Another factor that may have contributed to the species diversity on the scrubbers is the spatial heterogeneity of the systems. The scale of the systems is actually large, due to their 300’ length, and water flowing over this scale dimension created a mixture of regular, longitudinal zones and irregular, scattered patches. This spatial heterogeneity offers more opportunities for species colonization than would an equivalent surface area of a uniformly distributed environment.

The vertical structure of the turf community may contribute to the species diversity of the systems. The character of the algal communities is dominated by filamentous species, more-or-less attached to the screen, that form a “canopy” over the turf. Most of the rest of the community are single-cell “understory” species, some epiphytic on the filaments but many others just growing unattached among the filaments. Thus, the multi-celled filaments themselves contribute to the diversity but they also may facilitate the presence of single-celled species.

Finally, there is a heterogeneity in time imposed on the scrubbers due to seasonal change in temperature and other factors. As with other ecological communities, a seasonal succession of species occur since different species are adapted to the different conditions of the seasons. Since the sampling of the turf communities spanned at least one annual cycle, the seasonal succession of species contributed to the diversity of the systems.

Understanding the causes of the high species diversity of the Muddy Run ATS deserves more study. The diversity seems to be higher than would be expected from any single natural algal community. A further question is whether or not the high diversity contributes to the high productivity of the ATS relative to natural communities as was discussed earlier. This kind of question illustrates the importance of studying the ecology of these systems in order to maximize their functioning in nutrient removal and biofuel feedstock production.

71

ACKNOWLEDGEMENTS

Mary Helen Marsh, past Manager of Exelon Corporation’s hydroelectric facilities on the Susquehanna River was a catalyst from the start to the finish of the ATS project. Thomas Jenkins of Exelon provided invaluable assistance with the construction and operation of the ATS at Muddy Run. Tim Goertemiller, of Living Ecosystems in Easton, Maryland, helped with set up of the aluminum ATS and his firm designed and contructed the wooden ATS.

LITERATURE CITED

Adey, W. and K. Loveland. 2007. Dynamic Aquaria: Building Living Ecosystems, 3 rd edition. Elsevier, Amsterdam.

Boynton, W. R., W. M. Kemp and C. W. Keefe. 1982. A comparative analysis of nutrients and other factors influencing estuarine phytoplankton production. Pp. 69-90. in: Estuarine Comparisons. V. S. Kennedy (ed.). Academic Press, New York, NY.

Cahoon, D. R. 1975. Net Productivity of Emergent Vegetation at Horn Point Salt Marsh. M. S. Thesis, University of Maryland, College Park, MD.

Harding, L. W., Jr., D. Degobbis and R. Precali. 1999. Production and fate of phytoplankton: Annual cycles and interannual variability. Pp. 131-172. in: Ecosystems at the Land-Sea Margin: Drainage Basin to Coastal Sea. American Geophyical Union.

Johnson, M. 1970. Preliminary Report on Species Composition, Chemical Composition, Biomass and Production of Marsh Vegetation in the Upper Patuxent Estuary, Maryland. Chesapeake Biological Laboratory, Solomons, MD. Reference No. 70-130.

Mulbry, W., S. Kondrad, C. Pizarro and E. Kebede-Westhead. 2008. Treatment of dairy manure effluent using freshwater algae: algal productivity and recovery of manure nutrients using pilot-scale algal turf scrubbers. Bioresource Technology 99:8137-8142.

Mulbry, W., P. Kangas and S. Kondrad. In press. Toward scrubbing the Bay: Nutrient removal using small algal turf scrubbers on Chesapeake Bay tributaries. Accepted for publication in Ecological Engineering.

Odum, H. T. 1956. Primary production in flowing waters. Limnology and Oceanography 1:102-117.

Sellner, K. G. and M. E. Kachur. 1987. Phytoplankton. Pp. 12-37. in: Ecological Studies in the Middle Reach of Chesapeake Bay. Springer-Verlag, Berlin.

72

Stevenson, J. C. 1988. Comparative ecology of submersed grass beds in freshwater, estuarine, and marine environments. Limnology and Oceanography 33:867-893.

Turner, R. E. 1976. Geographic variations in salt marsh macrophyte production: a review. Contributions in Marine Science 20:47-68.

Wass, M. and T. Wright. 1969. Coastal Wetlands of Virginia. Interim Report to the Governor and General Assembly. Special Report in applied Marine Science and Oceanographic Engineering No. 10. Virginia Institute of Marine Science, Gloucester Point, VA.

Table 1. Average nutrient removal efficiencies measured at mid-day during spring-fall 2009 (n=17). These data were calculated by dividing the output concentrations (at the bottom of the systems) by the input concentrations (at the top of the systems).

Nutrient component

Aluminum ATS

Wood ATS

N-NO3

18%

27%

P-PO4

17%

50%

Total N

28%

37%

Total P

9%

17%

73

Table 2. Water quantity data from the raceways at Muddy Run. Turnover time was calculated based on a raceway volume calculated by multiplying raceway length (9144 cm or 300 feet) by raceway width (30 cm or 1 foot) by water depth.

Raceway

liters/sec

Aluminum ATS

at 2% slope in

flow rate,

depth, cm

average water time, minutes

water turnover

2008

with full

flow

1.10

1.5

6.2

Aluminum ATS

 

at 2% slope in

2009

with split

flow

0.83

1.5

8.3

Wood ATS in

 

2009

at 1% slope

with split flow

0.73

2.9

18.2

Aluminum ATS

 

at 0.5% slope in

2009

with split

flow

0.83

3.7

20.4

 

74

Table 3a. Biomass production of the Aluminum ATS at 2% slope. Data are grams dry weight/m2/day. Numbers in parentheses are the months included in the seasons.

Season

vacuum harvestgreenwater

slough

total

Summer 2008

14.2

2.4

1.4

18.0

(6-8)

Fall 2008

6.2

4.2

0.5

10.9

(9-11)

Winter 2008-2009

---

---

---

---

(12-3)

Spring 2009

4.0

8.6

0.7

13.3

(4-5)

Summer 2009

11.6

4.6

1.4

17.6

(6-7)

Fall 2009*

6.6

6.3

0.7

13.6

(8-10)

Growing Season

Averages

7.3

5.8

0.9

14.0

(fall 08-summer 09)

(52%)

(42%)

(6%)

* at 0.5% slope

 

75

Table 3b. Biomass production of the Wooden ATS. Data are grams dry weight/m2/day. Numbers in parentheses are the months included in the seasons.

Season

vacuum harvestgreenwater

slough

total

Fall 2008

1.8

6.2

---

(10-11)

Winter 2008-2009

---

---

---

(12-3)

Spring 2009

1.3

10.3

---

(4-5)

Summer 2009

4.8

6.6

0.9

12.3

(6-8)

Fall 2009

4.2

6.1

0.2

10.5

(9-11)

Growing Season

Averages

3.4

7.7

0.6

11.7

(spring 09-fall 09)

(29%)

(66%)

(5%)

 

76

Table 4. Net primary production for the Muddy Run ATS raceways as measured by diurnal oxygen curve analysis. Data are in units of grams O2/m2/day.

Dates

aluminum ATS

wooden ATS

9/17/08-9/18/08

7.2

---

10/29/08-10/30/08

2.5

3.2

12/18/08-12/19/08

1.5

0.8

4/7/09-4/8/09

1.1

1.0

6/11/09-6/12/09

2.4

3.8

7/30/09-7/31/09

8.6

6.8

8/13/09-8/14/09

6.5

6.4

9/25/09-9/26/09

8.2

7.4

11/12/09-11/13/09

3.0

2.3

77

Table 5. Comparison of growing season productivities for the ATS systems at Muddy Run and various natural aquatic plant communities of the Chesapeake Bay. Data for natural communities have been converted from various units to grams dry weight/m2/day, according to the footnotes given at the bottom of the table.

Plant community type

Net productivity,

g dry weight/m2/day

Reference

Aluminum ATS at Muddy Run

14.0

this study

Wooden ATS at Muddy Run

11.7

this study

Submerged aquatic vegetation* 28

Stevenson 1988

Emergent marshes**

11.3

Wass and Wright 1969

8.8

Johnson 1

4.9

Cahoon 1975

3.1

Turner 1976

Phytoplankton***

2.5

Selner and Kachur 1987

2.0

Boynton et al. 1982

1.5

Harding et al. 1999

* calculated assuming dry weight is 50% carbon.

** calculated assuming that annual belowground net production equals annual aboveground net production and assuming a 9 month growing season.

*** calculated assuming dry weight is 50% carbon.

78

Figure 1. Water temperature recorded at the top and bottom of the aluminum ATS over

Figure 1. Water temperature recorded at the top and bottom of the aluminum ATS over the study period.

79

Figure 2. Summary of production and nitrogen uptake on the aluminum ATS for 2009 80
Figure 2. Summary of production and nitrogen uptake on the aluminum ATS for 2009 80
Figure 2. Summary of production and nitrogen uptake on the aluminum ATS for 2009 80

Figure 2. Summary of production and nitrogen uptake on the aluminum

ATS for 2009

80

Figure 3. Summary of production and phosphorus uptake on the aluminum ATS for 2009 81
Figure 3. Summary of production and phosphorus uptake on the aluminum ATS for 2009 81
Figure 3. Summary of production and phosphorus uptake on the aluminum ATS for 2009 81

Figure 3. Summary of production and phosphorus uptake on the aluminum

ATS for 2009

81

Figure 4. Cumulative taxonomic species richness of the aluminum ATS over the study period. 82

Figure 4. Cumulative taxonomic species richness of the aluminum ATS over the

study period.

82

Appendix

C

Ozark Highland Algal Turf Scrubber:

Project Report

December 4, 2009

Prepared by:

The Center for Agricultural and Rural Sustainability University of Arkansas Division of Agriculture 203 Engineering Hall Fayetteville, AR 72701

83

TABLE OF CONTENTS

I. Introduction

1

II. Monitoring Strategy

1

a. Water Quality Analysis

b. Biomass analysis

c. Weather Monitoring

III. Data and Results

2

a. Dry Weight Data

2

b. Water Quality Analysis

4

c. Weather Data

10

IV. Analysis

11

V. Conclusions

14

VI. Appendix A: Anion Data

a. Orthophosphate

i

b. Nitrate-nitrite

iii

VII. Appendix B: Pictures

xxi

84

LIST OF FIGURES AND TABLES

Table 1: ATS Biomass Productivity Rates by Section and Averaged by Date

3

Table 2: Average Temperature Data by Deployment for the ATS System

5

Table 3: Average Dissolved Oxygen Data by Deployment for the ATS System

6

Table 4: Average pH Data by Deployment for the ATS System

6

Table 5: Average Specific Conductivity Data by Deployment for the ATS System

7

Table 6: Total Nitrogen Concentrations Above and Below the ATS System, with Percent

Removal

7

Table 7: Total Phosphorus Concentrations Above and Below the ATS System, with Percent

Removal

8

Table 1A: Orthophosphate Concentrations Above and Below the ATS System, with Percent

Removal

i

Table 2A: Nitrate-Nitrite Concentrations Above and Below the ATS System, with Percent

Removal

iii

Figure 1: Average Biomass Production Rates for Each Harvest Period. The line is the seven

day moving average, and the bars represent upper and lower 90 percent confidence intervals.

85

2

Figure 2: ATS System Growth Profile from the Upper to Lower Section for Harvest Date

August 24, 2009

3

Figure 3: Dissolved Oxygen Data: August 19, 2009 August 24, 2009

4

Figure 4: pH Data: August 19, 2009 August 24, 2009

4

Figure 5: Total Phosphorus Percent Removal from the ATS System

8

Figure 6: Total Nitrogen Percent Removal from the ATS System

9

Figure 7: Daily Orthophosphate Removal Across Two Harvest Periods at the ATS

9

Figure 8: 2009 Precipitation Data Collected Daily for Springdale, AR

10

Figure 9: 2009 Maximum and Minimum Ambient Temperature Data Collected Daily for

Springdale, AR

10

Figure 10: Biomass Production and Orthophosphate Removal Over the 9-month Harvest

Period at the ATS

11

Figure 11: Biomass Production and Nitrate Removal Over the 9-month Harvest Period at the

ATS

11

Figure 12: Biomass Production and Total Phosphorus Removal Over the 9-month Harvest

Period at the ATS

12

Figure 13: Biomass Production and Total Nitrogen Removal Over the 9-month Harvest

Period at the ATS

86

12

Figure 14: Biomass Production and Maximum Ambient Temperature Over the 9-month

Harvest Period at the ATS

13

Figure 15 Biomass Production and Average Precipitation Over the 9-month Harvest Period at

the ATS

13

Figure 1A: Orthophosphate Removal Over the 9-month Harvest Period at the ATS

ii

Figure 2A: Nitrate-Nitrite Removal Over the 9-month Harvest Period at the ATS

iv

87

INTRODUCTION

The University of Arkansas Center for Agricultural and Rural Sustainability (CARS) designed and constructed an algal turf scrubber (ATS) in the Ozark Highland Region as part of a project scope of work with the Smithsonian Institute. CARS operated and monitored the system for one year. Construction began in May of 2008, and was completed in November of the same year. However, due to a combination of inclement weather and equipment and power failures, the system was not operating continuously until March 1, 2009. This report summarizes the biomass production, water quality, and weather data collected during the nine month operating time.

MONITORING STRATEGY

The sampling regime consisted of weekly monitoring of the system biomass production and water quality. The sample regime included water quality analysis, biomass analysis, and weather monitoring.

Water Quality Analysis

A set of multiple parameter water quality monitoring devices (data sondes) were used to monitor

pH, dissolved oxygen, specific conductivity, and water temperature. The data sondes took

measurements every 10 minutes during the deployment period. In addition to the continuous monitoring of these parameters, water samples were taken every harvest period from the inside

of the surger at the intake, and from the sampling reservoir at the outflow. The water samples

were frozen and then analyzed for orthophosphate and nitrate-nitrite using ion chromatography,

as well as total phosphorus and total nitrogen at the University of Arkansas. In addition to the weekly sampling events, a set of samples were taken every day for 14 days during the month of July to establish the changes in water quality and nutrient removal across harvest periods.

Biomass Analysis

The algal biomass was harvested every week using a standard wet/dry shop vac. The algae was harvested from three 10-foot sections at the upper, middle, and lower sections of the flow-way, and then sent to the Ecological Engineering Laboratory at the University of Arkansas for dry weight analysis. The dry weight was determined by drying each sample at 105°C for 24 hours. From this, the dry weight per unit of area produced by the system was determined. The slough coming off the system was also collected throughout the harvest period, and the dry weight was determined along with the algae samples taken from the harvest. In addition, a ten centimeter square section of mesh was removed periodically for taxonomic analysis.

Weather Monitoring

Weather data was collected through local weather monitoring stations. Precipitation, maximum and minimum temperatures, average humidity, and dew point were recorded for each day.

88

DATA AND RESULTS

DRY WEIGHT ANALYSIS

Algal biomass was collected from three sections on the flow-way from March 2009 through August

2009. The productivity rates determined for each section (and adjusted for the slough yield per unit area) are listed in Table 1. The average productivity over time is shown in Figure 1. In addition to repeated sampling at the upper, middle, and lower sections, the amount of variation in growth across the entire

length of the flow-way was also measured.

On August 24, 2009 the system was harvested at alternating

10-foot intervals down the flow-way.

A plot of the system profile is shown in Figure 2. It is important

to note that three major system shutdowns occurred during the biomass sampling period. The first shutdown was in early May, and was the result of theft/vandalism. The second shutdown occurred at the

end of May, and was the result of a power failure associated with the incident that occurred earlier in the

month.

treatment plant. This situation also coincided with heavy rains, and resulted in a lack of data for the first half of October. In addition to the system shutdowns, there were two instances of irregular harvest procedure, the first on July 2 nd , and the other on August 4 th . On these days, thunderstorms interrupted the

harvest session, and the harvest was halted after collecting the biomass samples for dry weight analysis. The following day, the entire flow-way was vacuumed to ensure that there was no irregularity in the growing time across the trough.

The third occurred in early October, and was the result of a power failure at the wastewater

90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 3/1/2009 4/20/2009 6/9/2009 7/29/2009 9/17/2009
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
3/1/2009
4/20/2009
6/9/2009
7/29/2009
9/17/2009
11/6/2009
Average Production Rates (dw g/sq m/day)

Figure 1: Average Biomass Production Rates for Each Harvest Period. The line is the seven day moving average, and the bars represent upper and lower 90 percent confidence intervals.

89

   

Production (g/sq m/day)

 

Date

# Days

Upper

Middle

Lower

Average

3/10/2009

10.00

9.99

3.60

 

6.80

3/17/2009

7.00

9.32

9.52

9.97

9.60

3/30/2009

13.00

22.45

21.70

21.26

21.81

4/11/2009

12.00

21.35

9.72

18.98

16.69

4/16/2009

5.00

40.40

17.66

31.66

29.91

4/23/2009

7.00

4.69

5.96

15.98

8.88

5/23/2009

7.00

11.83

8.70

16.14

12.22

6/9/2009

9.00

17.41

13.52

22.75

17.89

6/18/2009

9.00

37.17

14.26

55.99

35.81

6/27/2009

9.00

36.78

30.30

36.08

34.39

7/2/2009

5.00

94.56

17.88

42.88

51.77

7/7/2009

5.00

35.22

16.08

15.47

22.25

7/11/2009

4.00

40.79

22.75

23.13

28.89

7/15/2009

4.00

46.19

14.57

18.92

26.56

21 19 17 15 13 11 9 7 5 0 50 100 150 200 250
21
19
17
15
13
11
9
7
5
0
50
100
150
200
250
300
Algal Density ( g dw/ft 2 )

Location on Floway (ft)

Figure 2: ATS System Growth Profile from the Upper to Lower Section for Harvest Date August 24, 2009

90

WATER QUALITY ANALYSIS

The dissolved oxygen, pH, temperature, and conductivity levels were monitored daily at the system inflow and outflow. A total of 14 successful one-week data sonde deployments were performed from March through November.

Figures 3 and 4 show pH and dissolved oxygen percent saturation data recorded during the harvest period from August 19, 2009 through August 24, 2009. The trends shown in these graphs are typical of pH and dissolved oxygen levels measured during a harvest period.

dissolved oxygen levels measured during a harvest period. 300 250 200 150 100 50 0 8/19/2009
300 250 200 150 100 50 0 8/19/2009 8/20/2009 8/21/2009 8/22/2009 8/23/2009 8/24/2009 8/25/2009 Outflow
300
250
200
150
100
50
0
8/19/2009
8/20/2009
8/21/2009
8/22/2009
8/23/2009
8/24/2009
8/25/2009
Outflow
Inflow
DO Saturation (%)
8/24/2009 8/25/2009 Outflow Inflow DO Saturation (%) Figure 3: ATS System Dissolved Oxygen Data: August 19,

Figure 3: ATS System Dissolved Oxygen Data: August 19, 2009 August 24, 2009

pH

10.5 10 9.5 9 8.5 8 7.5 7 8/19/2009 8/20/2009 8/21/2009 8/22/2009 8/23/2009 8/24/2009 8/25/2009
10.5
10
9.5
9
8.5
8
7.5
7
8/19/2009
8/20/2009
8/21/2009
8/22/2009
8/23/2009
8/24/2009
8/25/2009
Outflow
Inflow

Figure 4: ATS System pH Data: August 19, 2009 August 24, 2009

91

For each parameter the maximum and minimum values, along with the averages and standard deviations, from each data sonde deployment are given in Tables 2 through 5.

   

Temperature

 
 

Upper

   

Lower

 

Summary

Deployment #

Deployed

Retrieved

Max

Min

Deployment # Deployed Retrieved Max Min S.D. Max Min S.D. Δ x min Δ x max