Вы находитесь на странице: 1из 12

Journal of Applied Phycology 15: 325–335, 2003.

325
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Physiological characteristics of cyanobacteria in pulp and paper


waste-treatment systems

A.E. Kirkwood 1,2,*, C. Nalewajko 1 and R.R. Fulthorpe 1


1
Division of Life Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto, M1C 1A4,
Ontario, Canada; 2Current address: Department of Botany, Oklahoma State University, 104 LSE, Stillwater,
Oklahoma 74078, USA; *Author for correspondence (e-mail: kirkwoa@okstate.edu; phone: +1-405 744 5956)
Received 3 November 2002; accepted in revised form 24 January 2003

Key words: Biodegradation, Cyanobacteria, Heterotrophy, Mixotrophy, Photosynthesis, Pulp and paper waste-
treatment

Abstract

An investigation was made into the physiological characteristics of the extensive cyanobacterial communities in
pulp and paper secondary waste-treatment systems, including the capacity of isolates to biodegrade organic con-
taminants in these systems. Although pulp and paper waste-treatment systems were found to be severely light-
limited, photosynthesis-irradiance curves indicated that shade-adapted cyanobacterial communities could fix
conspicuous amounts of inorganic carbon via photosynthesis. Of 21 cyanobacterial strains isolated from pulp and
paper waste-treatment systems located in 4 countries, all except one were capable of glucose uptake in the light,
and 19 also showed uptake in the dark. In the 6 species tested, glucose and acetate addition stimulated growth in
low light in all except one species. Aphanocapsa rivularis grew equally well on glucose and acetate in the dark
but Pseudanabaena sp. grew well on acetate, and minimally on glucose. Growth stimulation by glucose and
acetate in these two strains was greater in low than in high light. Pseudanabaena sp. and Phormidium animale
both accumulated 2,4-dichlorophenol and 3-chlorobenzoate but showed minimal mineralization to CO 2. None of
the four species tested could accumulate or degrade phenol or dichloroacetate. It is concluded that, depending on
the light conditions, cyanobacteria contribute organic carbon in photosynthesis, and/or remove small organic
molecules during mixotrophic and heterotrophic growth but are not important degraders of contaminants in these
waste treatment systems.

Introduction to several days. Both ASBs and ASs are open-air fa-
cilities. The ASBs are comparable to large intercon-
The pulp and paper industry depends on secondary necting ponds or small lakes (e.g., total volume = 1.5
(biological) waste-treatment systems to treat highly million m 3). Conventionally, these waste-treatment
concentrated organic waste. Bleached-kraft mill systems have been viewed as heterotrophic, but the
wastes contain hundreds of wood extractives (e.g., recently documented ubiquitous presence of cyano-
resin acids) and chlorinated organic compounds (e.g., bacteria in the microbial communities (Kirkwood et
chlorophenolics) of environmental concern. Conse- al. 2001) raises the question of their role in these sys-
quently, pulp and paper wastewater is toxic and re- tems.
quires microbial mineralization to reduce effluent tox- Photosynthetic eukaryotes are absent from ASBs
icity. Aerated stabilization basins (ASBs) and suggesting that this environment is not ideal for pho-
activated sludge systems (ASs) used by the pulp and tosynthetic organisms yet Kirkwood et al. (2001) doc-
paper industry for secondary waste-treatment are de- umented the existence of dynamic cyanobacterial
signed to support high densities of heterotrophic bac- communities some of which exceeded the biomass of
teria over a hydraulic retention time of several hours heterotrophic bacteria. Regardless of geographical lo-
326

cation Phormidium, Geitlerinema and Pseudana- national Equipment Co., USA) and resuspended in
baena were the three dominant taxa in ASBs from equal volumes of modified BG-11 medium (Allen
Brazil, Canada, New Zealand, and USA. 1968). BG-11 was modified by reducing the sodium
Although all cyanobacteria are photoautotrophic, nitrate concentration to one-tenth the usual concentra-
many can utilize simple dissolved organic carbon tion, removing all organic components (citric acid,
(DOC) compounds for heterotrophic growth or for ferric ammonium citrate, and sodium EDTA) and re-
mixotrophic growth in the light. (Khoja and Whitton placing the iron with 3 µg L −1 ferric chloride. 400-mL
1971; Fogg et al. 1973; Sahu and Adhikary 1982; aliquots of the resuspended samples were dispensed
Radwan and Al-Hasan 2000). Waters in ASBs are into polycarbonate bottles with screw-caps and placed
highly coloured, and, in spite of mechanical agitation, in series in a photosynthesis-irradiance box with tem-
are likely to provide a severely light-limited environ- perature control. A 500-W quartz-halogen lamp (Phil-
ment in which small molecules such as glucose and lips 12013 R) was placed at the front of the box and
acetate present in the wastewater may be important irradiance measurements were taken with a Model
for cyanobacterial growth. Cyanobacteria also have QSL-100 quantum meter. Each bottle was spiked with
the potential for catabolism of the contaminants in 200 µL of 20 µCi mL −1 14C-NaHCO 3, specific activ-
wastewater. A few studies have shown that certain ity 52.9 µCi µmol −1 (Vollenweider 1969). One-mL
cyanobacterial taxa are capable of biodegrading hy- sub-samples were taken from each bottle and dis-
drocarbons (Narro 1987; Narro et al. 1992; Shash- pensed into scintillation vials with 0.5 mL of phen-
irekha et al. 1997; Radwan and Al-Hasan 2000) and ethylamine for measurements of the total radioactiv-
the highly chlorinated aliphatic pesticide lindane (Ku- ity added. Radiolabeled samples were incubated for 4
ritz and Wolk 1995). hours at 28 °C. At the termination of the experiment,
The aim of this study was to investigate the meta- 50-mL sub-samples from each bottle were filtered on
bolic characteristics of cyanobacteria in pulp and pa- Whatman G/FC filters and dissolved in 15 mL of
per waste-treatment systems in order to gain a better Ecolume scintillation fluor. Radioactivity was mea-
understanding of their physiological contributions sured in a Beckman Model LS-6800 scintillation
during biological waste-treatment. This in turn would counter.
provide some valuable insight into the role of cyano- To assess organic carbon uptake by cyanobacteria,
bacteria in the microbial community as well as their 50 µL from 100 mM glucose stock solution with 12
potential impact on waste-treatment efficiency. µCi mL −1 of D-[U- 14C] glucose were added to 20 mL
of early exponential-phase cultures of axenic cyano-
bacteria grown in the previously described organic-
Materials and methods free BG-11. Triplicate cultures were fixed with form-
aldehyde (2% v/v) prior to radioisotope addition to
Data on environmental variables from the Idaho, New correct for any abiotic 14C-glucose adsorption (Paerl
Zealand, and Maine mills were provided by mill tech- et al. 1993). Both live and fixed cultures were incu-
nicians and were based on weekly measurements us- bated in triplicate in the light (46.3 µmol m −2 s −1) and
ing standard protocols (American Water Works Asso- dark at 26 °C for 4 h. After incubation each culture
ciation 1995). Light extinction coefficients and was filtered on a 2-cm diameter Whatman G/FC filter
irradiance depth profiles in the ASBs were based on and rinsed three times with double-distilled water.
the irradiance measurements of an immersible Model The radioactivity of filters was measured as described
QSL-100 quantum meter (Biospherical Instruments, for 14C photosynthesis experiments.
USA). Photosynthesis as a function of irradiance To measure the ability of cyanobacteria to biode-
(photon flux density) was measured in the laboratory grade organic contaminants found in pulp and paper
using freshly collected water samples from the Maine effluent, 50 µL 14C-2,4-dichlorophenol (0.1 µCi µL −1)
ASB at the mid-point of treatment, and from the aer- and 14C-chlorobenzoate (0.1 µCi µL −1) from 100 mM
ated basin, the mid-point of treatment in the AS sys- stock solutions were added to borosilicate bottles con-
tem used at the Quebec 2 mill. taining 50 mL of early exponential phase axenic cul-
To eliminate the interference of highly-coloured tures grown in modified, organic-free BG-11. Bottles
substances in wastewater with the 14C-uptake, cyano- with only sterile BG-11 medium were also spiked
bacteria were collected from the wastewater samples with radioisotope to serve as controls. 1 mL sub-sam-
by centrifugation (8000 × g, HN-S centrifuge, Inter- ples were taken from each bottle for measurement of
327

total radioactivity. To assess 14CO 2 release from con- concentration of 1.5 mM. Flasks without nitrogen ad-
taminant mineralization, sterile filter paper strips pre- dition (control), with sodium nitrate addition, and am-
viously treated with 1-M NaOH solution were hung monium sulfate addition were incubated in triplicate
from the lip of each bottle and clamped on with a in the light (46 µmol m −2 s −1) at 28 °C for 5 days.
rubber stopper and aluminum seal. Radiolabeled cul- Phosphate uptake kinetics was assessed in freshly
tures and controls were grown in the light (46.3 collected wastewater samples from Maine. Carrier-
µmol m −2 s −1) and dark at 26 °C for 7 days. At the free 32P-PO 4 was added and the radioactivity remain-
termination of each biodegradation experiment, filter ing in the filtrates was measured at 5 to 20-minute
paper strips were dissolved in 2 mL of Ecolume scin- intervals for 30 minutes. The samples were incubated
tillation fluor. Cyanobacterial biomass was collected at 26 °C and were illuminated by high-output fluo-
on G/FC filters, rinsed three times with double-dis- rescent lights. Radioactivity in filtrates was measured
tilled water, and dissolved in 15 mL of Ecolume. Ra- as previously described (Lean and Nalewajko 1979).
dioactivity of filters with biomass and trapped 14CO 2 To assess the autotrophic, mixotrophic and het-
was measured as previously described. erotrophic growth potential of cyanobacteria, sterile
To assess phenol biodegradation, 100 µL of phe- flasks with 50 mL of modified, organic-free BG-11
nol stock was added to 20 mL of early exponential- were divided into three treatments: unamended (con-
phase cyanobacteria grown in modified, organic-free trol), glucose-addition (1 mM final concentration),
BG-11 for a final concentration of 1-mM phenol. Cul- and acetate-addition (3 mM final concentration). Trip-
ture flasks were placed in triplicate in the light (46.3 licate flasks of each treatment were inoculated with 1
µmol m −2 s −1) and dark, and incubated at 26 °C for 5 mL of axenic late exponential-phase culture and
days. The amount of phenol remaining in each flask placed in high light (46.3 µmol m −2 s −1), low light (8
was monitored over time by taking 1-mL subsamples µmol m −2 s −1) and dark incubators at 26 °C for 5
and centrifuging (13,000 × g, Micro Biofuge, Heraeus days. In Aphanocapsa rivularis PP19 and Pseudana-
Instruments, USA) for 10 minutes. The supernatant baena PP16, which grew homogeneously in suspen-
from each tube was transferred into plastic-stoppered sion, yields were measured daily as optical density at
glass cuvettes and immediately frozen. All samples 550 nm using a Spectronic (Genesys 5) spectropho-
for each experimental set were thawed at the same tometer (Milton Roy, USA). In all species, cultures
time and analyzed by high pressure liquid chromato- were filtered at the end of each experiment and bio-
graphy (Waters Corporation, USA) with a 70:30 mix- mass was measured as chlorophyll a.
ture of analytical-grade 1% phosphoric acid and ace- For chlorophyll a analysis, the samples were fil-
tonitrile run at 1 mL min −1 using a NovaPak C18 tered on Whatman GF/C filters, frozen for 48 h, and
reverse-phase column. the chlorophyll extracted with 20 mL of 90% analyti-
To assess dichloroacetate biodegradation, 100 µL cal-grade acetone. Acetone extracts were centrifuged
of neutralized dichloroacetate was added to 20 mL of for 10 min (8000 × g, HN-S centrifuge, International
early exponential phase cyanobacteria grown in mod- Equipment Co., USA) and chlorophyll a in the super-
ified, organic-free BG-11, for a final concentration of natant analyzed in a Spectronic Genesys 5 spectro-
2.5-mM dichloroacetate. The medium was further photometer (Milton Roy, USA) according to Hans-
modified to reduce background chloride by replacing man (1973).
ferric chloride with ferric ammonium sulfate (6
mg L −1). Culture flasks were placed in triplicate in the
light (46.3 µmol m −2 s −1) and dark, and incubated at Results
26 °C for 5 days. Dichloroacetate biodegradation was
measured as the amount of chloride ion produced by Secchi disk depths were low and light extinction co-
dechlorination at the end of the experiment. Chloride efficients high, especially at the Quebec 2 site (Table
concentration was measured in 5-mL subsamples 1, Figure 1). The photosynthesis-irradiance curves
from each replicate using a Fisher Scientific Accumet (Figure 2) for samples from Quebec 2 show lower
1003 ion-probe. light-saturated rate of photosynthesis (P max = 0.104
To determine the impact of combined nitrogen ad- µg C µg Chl a h −1), light-limited photosynthesis (␣ =
dition on cyanobacterial biomass, 200 mL of waste- 0.013), and light-saturation (I k = 6.7 µmol m −2 s −1)
water in sterile 250-mL flasks was amended with am- than the Maine communities (P max =
monium or nitrate to yield a total nitrogen
328

Table 1. Light extinction coefficients and Secchi disk depths in


four pulp and paper secondary waste-treatment systems.

Mill Location Extinction coefficient Secchi Disk depth


(␩ m −1) (m)

Brazil 6.46 0.30


Idaho 6.97 0.24
Maine 6.89 0.25
Quebec 2 9.16 0.19

Figure 2. Photosynthesis-irradiance curves for cyanobacterial


communities in the Maine and Quebec 2 secondary waste-treat-
ment systems.

the Maine wastewater (Figure 4) indicates a moder-


ately P-limited system.
All 21 cyanobacterial taxa isolated from various
pulp and paper waste-treatment systems could take up
glucose in the light (Table 3) and all but two also took
up glucose in the dark. With the exception of 4 spe-
cies, uptake in the light was much greater than in the
dark. With the exception of P. animale PP1, glucose
Figure 1. Irradiance-depth profiles for the Maine and Quebec 2
secondary waste-treatment systems.
and acetate addition significantly (Tukey-Kramer, p <
0.05) increased final chlorophyll a concentrations in
0.214 µg C µg Chl a h −1, ␣ = 0.021, I k = 7.35 high light. In low light, responses to each organic
µmol m −2 s −1). substrate differed among the species (Figure 5). Final
Environmental variables at the Idaho, New biomass of Aphanocapsa conferta PP18 and T. bour-
Zealand and Maine mills were comparable, with the rellyi PP17 increased significantly (Tukey-Kramer, p
exception of biological oxygen demand (BOD), < 0.05) in the presence of glucose compared to ac-
which was an order of magnitude lower in Maine, and etate. Conversely, the biomass of A. rivularis PP19
total inorganic nitrogen (TIN), which was two orders was significantly (Tukey-Kramer, p < 0.05) greater on
of magnitude higher in Maine (Table 2). acetate than on glucose. Both glucose and acetate sig-
The effects of combined nitrogen addition on chlo- nificantly (Tukey-Kramer, p < 0.05) increased the
rophyll a differed among mills, even though chloro- biomass of A. conferta PP18 and Pseudanabaena
phyll a concentrations in the controls were not signif- PP16, and neither organic substrate had a significant
icantly different (ANOVA, p > 0.05) (Figure 3). (ANOVA, p > 0.05) effect at low light intensity on
Ammonium and nitrate addition greatly decreased cy- the biomass of Cyanosarcina PP21 or Phormidium
anobacterial biomass in the wastewaters from Idaho animale PP1.
and New Zealand, respectively. Combined nitrogen Daily optical density measurements showed that at
addition significantly increased cyanobacterial bio- the mid-exponential phase of growth, glucose or ac-
mass in Maine’s wastewater, particularly when am- etate considerably increased the yield of Aphano-
monium was added. Phosphate turnover time of 1.7 h capsa rivularis PP19 and Pseudanabaena PP16. The
calculated from phosphate uptake data measured in stimulation was greater in low than in high light (Ta-
329

Table 2. Environmental variables in aerated stabilization basins (ASBs) used for secondary waste-treatment in Idaho, New Zealand and
Maine. All mills use elemental chlorine-free bleaching. NA = data not available.

Variable Idaho New Zealand Maine

Temperature (°C) 28.1 34.7 31


pH 7.7 7.0 7.3
Total Suspended Solids (mg L −1) 126 132 107
Colour (mg L −1) NA 347 308
Biological Oxygen Demand (mg L −1) 203 125 34.5
Total Inorganic N (mg L −1) < 0.10 < 0.10 2.2
Total P (mg L −1) 0.67 1.3 0.8

Figure 3. Effect of combined-nitrogen addition on cyanobacterial


biomass measured as chlorophyll a in three pulp and paper waste
treatment communities. Figure 4. Radiophosphorus uptake kinetics in the Maine second-
ary waste-treatment community.
ble 4). There was greater stimulation of Pseudana-
baena PP16 by acetate than by glucose both in high amounts of 14C-CBA in the light and dark, though
and low light, and increased yield on acetate than accumulation was lower in Pseudanabaena PP16.
glucose in the dark (Figure 6). Yields of A. rivularis Negligible amounts of 14C-CBA were mineralized in
PP19 in the dark was similar on glucose and acetate the light and dark by either species. None of the four
(Figure 6). cyanobacteria tested could biodegrade phenol or
Accumulation and mineralization of 2,4-dichlo- DCA in the light or dark.
rophenol (DCP) and 3-chlorobenzoate (CBA) differed
in Pseudanabaena PP16 and Phormidium animale
PP1. DCP accumulation by Pseudanabaena PP16 Discussion
was significantly (Student’s t-test, p < 0.05) greater
in the light than dark, but there was no significant In spite of the severe light attenuation and an euphotic
difference between light and dark treatments in P. an- zone confined to less than one meter in the wastewa-
imale PP1. Less than ten percent of total 14C-DCP ter treatment systems, we have measured relatively
was mineralized by Pseudanabaena PP16 in the light high photosynthetic rates and assimilation numbers in
and no mineralization was detected in the dark. In cyanobacterial communities from two of these sys-
contrast, no mineralization of 14C-DCP by P. animale tems. Photosynthesis-irradiance curves from the Main
PP1 was detected in the light, but approximately ten and Quebec 2 sites show that the cyanobacterial com-
percent was detected in the dark. Pseudanabaena munities were adapted to a low-light regime. The rel-
PP16 and P. animale PP1 accumulated similar
330

Table 3. Glucose uptake in the light and dark by axenic cyanobacteria isolated from pulp and paper waste-treatment systems. Standard errors
are presented in brackets.

Source Mill Genus cf Species Glucose uptake (µmol glucose µg Chl a h −1)

Strain ID Light Dark

New Zealand Aphanocapsa conferta PP18 2.3 (0.03) 1.76 (0.1)


New Zealand Aphanocapsa rivularis PP19 1.84 (0.05) 1.4 (0.01)
Manitoba, CAN Aphanothece floccosa PP20 0.37 (0.01) 0.48 (0.12)
Brazil Cyanosarcina sp. PP21 7.65 (0.09) 5.32 (0.12)
Idaho, USA Geitlerinema sp. PP12 0.03 (0.02) 0.07 (0.06)
New Zealand Geitlerinema sp. PP8 0.91 (0.02) 0.39 (0.02)
Maine, USA Geitlerinema amphibium PP13 0.02 (0.01) 0.01 (0)
Manitoba, CAN Geitlerinema amphibium PP14 0.01 (0) 0
New Zealand Geitlerinema carotinosum PP9 0.29 (0) 0.25 (0.01)
New Zealand Geitlerinema ionicum PP10 0.01 (0) 0
New Zealand Geitlerinema pseudacusitissimum PP11 0.03 (0.01) 0.01 (0)
Alberta 1, CAN Lyngbya sp. PP15 0.44 (0.11) 0.22 (0.05)
Maine, USA Phormidium animale PP1 2.78 (0.02) 2.36 (0.03)
Maine, USA Phormidium autumnale PP2 0.04 (0.01) 0.03 (0)
Idaho, USA Phormidium deflexoides PP5 0.11 (0.01) 0.18 (0.04)
Brazil Phormidium insigne PP7 0.17 (0.02) 0.27 (0.02)
Maine, USA Phormidium rimosum PP3 2.78 (0.04) 2.5 (0.04)
Quebec 2, CAN Phormidium rimosum PP4 0.02 (0) 0.01 (0)
Quebec 1, CAN Pseudanabaena sp. PP16 1.43 (0.01) 0.85 (0.01)
Alberta 2, CAN Radaisia sp. PP22 0.18 (0) 0.09 (0)
New Zealand Tychonema bourrellyi PP17 9.37 (0.09) 8.6 (0.19)

atively high ␣ and low I k values, and the low P max, organisms. Cyanobacteria in pulp and paper waste-
especially in the Quebec 2 community, indicate a de- treatment systems likely depend on wavelengths of
creased photosynthetic capacity normally associated 600–700 nm by utilizing both chlorophyll a and the
with shade adaptation (Beardall and Morris 1976; accessory pigment phycocyanin for photosynthetic
Falkowski 1984). energy (Chapman 1973; Wall and Briand 1979). The
Although light penetration is low in these systems, blue-green pigment phycocyanin was clearly evident
the continuous mechanical mixing would ensure pe- during the extraction of chlorophyll a from pulp and
riods of exposure to light for the cyanobacterial com- paper wastewater communities.
munity. In natural systems, constant yet stabilized Even with the light limitation imposed on cyano-
mixing of the water column normally results in low bacteria in these waste-treatment systems, they are
I k, but relatively high P max, values for resident pho- important members of the microbial community
tosynthetic communities (Harris et al. 1980). Thus, (Kirkwood et al. 2001). The widespread capacity we
stabilized vertical mixing in pulp and paper wastewa- documented among the cyanobacterial isolates to take
ters may be an important factor in promoting higher up glucose and acetate and grow mixotrophically
cyanobacterial biomass levels than would be expected and/or heterotrophically, no doubt contributes to their
in these light limited systems. success in these systems. In particular, the enhance-
Photosynthesis depends not only on available light ment of growth by glucose and acetate at low light is
intensity, but also on spectral distribution. Coloured a useful feature in these light-limited systems. With
DOC tends to absorb photosynthetically available ra- ample amounts of simple DOC compounds available
diation (PAR) at the lower wavelengths, 400–500 nm in pulp and paper wastewater (Hausalo 1995; Liukko
(Wetzel 1983). Highly coloured waters such as pulp and Poppius 1999), it is not surprising that cyanobac-
and paper wastewater would absorb and restrict the teria from this environment have the capacity to uti-
availability of these wavelengths for photosynthetic lize at least some of these organic compounds. None
331

Figure 5. Final chlorophyll a values for cyanobacteria grown under photoautotrophic and mixotrophic conditions. The control represents
chlorophyll a from cultures grown in mineral medium. Taxonomic names for the cyanobacteria are shown in Table 3.

Table 4. Growth at the mid-exponential stage in high and low-light in the presence of glucose or acetate. Growth was measured as optical
density (O.D.) and growth in mineral medium served as the control.

Strain % control

Glucose (1 mM) Acetate (3 mM)

high light low light high light low light

Aphanocapsa rivularis PP19 252 395 137 375


Pseudanabaena PP16 150 200 215 346

of the 21 cyanobacterial isolates were found to be (Fogg et al. 1973). The preferential use of acetate
obligate photoautotrophs, which suggests that the over glucose by Pseudanabaena PP16 is thus unusual
low-light, high DOC environment of pulp and paper and merits further investigation.
waste-treatment systems has selected for facultative It is desirable to have high levels of nitrogen and
organotrophs. Although some cyanobacteria are obli- phosphorus in pulp and paper secondary waste-treat-
gate phototrophs, among the heterotrophs, glucose ment systems to maintain dense heterotrophic bacte-
and other sugars in general are the most frequently rial populations. However, it is not uncommon for
used substrates. Acetate and other Krebs Cycle inter- pulp and paper wastewaters to be low in combined
mediates typically do not support growth in the dark inorganic nitrogen (Bryant et al. 1997; Jarvinen
332

from other sources. The suppressed cyanobacterial


growth response to ammonium and nitrate addition
observed in this study could be a result of inhibition
of nitrogen fixation by the addition of combined ni-
trogen (Stewart 1964). Several authors have estab-
lished that nitrogen fixation can occur in pulp and
paper waste-treatment systems, including the New
Zealand site (Gapes et al. 1999; Gauthier et al. 2000).
Because nitrogen fixers have lower affinities for com-
bined nitrogen uptake (Reuter et al. 1986; Pinckney
et al. 1995), the competitive advantage within the mi-
crobial community may have shifted to non-nitrogen
fixers upon combined-nitrogen addition. Het-
erotrophic bacteria tend to be more efficient at inor-
ganic nutrient uptake than photosynthetic microor-
ganisms (Currie and Kalff 1984; Currie 1990; Jansson
1993) and thus may have outcompeted the cyanobac-
teria.
Most likely, the cyanobacterial community from
Maine was adapted to high levels of inorganic nitro-
gen from ammonium nitrate fertilization practices,
and with additional inputs of nitrogen in our experi-
ments, cyanobacteria reacted with an increase in bio-
mass. Therefore, our results indicate that cyanobacte-
rial growth remains nitrogen-limited, even with
routine wastewater fertilization. Phosphate uptake ki-
Figure 6. Growth of Aphanocapsa rivularis PP19 and Pseudana- netics in wastewater from Maine also indicated mod-
baena PP16 in the presence of glucose and acetate in the dark. The erate P limitation in a system with high total P con-
control represents growth in mineral medium.
centrations. Considering the large densities (10 7–10 10
cells mL −1) of bacteria in pulp and paper wastewater
1997). In common with many pulp and paper waste- communities, these results suggest that inputs of ni-
treatment systems, two of the mills in this study (Ida- trate, ammonium and phosphate are offset by high
ho and New Zealand) had non-detectable levels of microbial demand for these nutrients.
inorganic nitrogen. In contrast, concentrations of in- In contrast to other studies (Kuritz and Wolk 1995;
organic nitrogen were higher at the Maine site be- Shashirekha et al. 1997), cyanobacteria from pulp and
cause ammonium and nitrate are added on a daily paper waste-treatment systems in the present study
basis to the waste-treatment system. It is interesting did not substantially mineralize the contaminants
that cyanobacterial biomass was comparable among used. Less than 10% of 2,4-dichlorophenol was min-
mills regardless of the nitrogen status of the waste- eralized by cyanobacteria over the course of an aver-
water, yet growth responses to ammonium or nitrate age hydraulic retention time. Compared to het-
addition varied. There was no biomass increase fol- erotrophic bacteria from these systems (Puhakka et al.
lowing ammonium or nitrate addition to sites with no 1994; Fulthorpe and Allen 1995; Liss et al. 1997),
detectable N. In contrast, biomass in wastewater from cyanobacteria would not be important contributors to
Maine significantly increased with combined nitrogen microbial biodegradation of contaminants during pulp
addition. These results indicate that each cyanobacte- and paper secondary waste-treatment. However, as
rial community was adapted to the nitrogen status of with other chlorinated organic compounds (Twiss et
its environment. al. 1999; Nikkila et al. 2001), cyanobacteria accumu-
The presence of relatively large cyanobacterial lated large amounts of 2,4-dichlorophenol and to a
biomass in the Idaho and New Zealand wastewaters lesser extent, chlorobenzoate by adsorbtion or absorp-
with negligible inorganic nitrogen concentrations sug- tion. This has important implications for the fate of
gests that the cyanobacteria were acquiring nitrogen these compounds during secondary treatment, be-
333

Figure 7. Accumulation and mineralization to CO 2 of radiolabeled 2,4-dichlorophenol (DCP) and 3-chlorobenzoate (CBA) by axenic cul-
tures of P. animale PP1 and Pseudanabaena PP16 after a 7-day incubation period in the light and dark.

cause the contaminants would be removed from distinct possibility. Cyanobacterial utilization of or-
wastewater, and would not be bioavailable for degra- ganic compounds would contribute to the desired re-
dation by the heterotrophic community. Therefore the duction of BOD, but possibly at the expense of or-
fate of these contaminants may be determined in part ganic contaminant biodegradation. Many organic
by the fate of cyanobacterial biomass in secondary contaminants are co-metabolized by bacteria and re-
waste-treatment facilities. quire an additional substrate(s) for growth (Schmidt
The input of organic carbon from cyanobacterial et al. 1985). Cyanobacterial utilization of substrates
photosynthesis into a system designed to remove or- such as glucose and acetate would reduce their avail-
ganic carbon is undesirable. However, organic carbon ability to the bacterial degraders.
contribution by cyanobacteria (including carbon fixed Considering the negligible role that cyanobacteria
plus biomass) at two of the study sites was less than would have in community biodegradation of waste-
10% of the total biological oxygen demand in the ef- water contaminants, it would seem that cyanobacteria
fluent (Table 2). We conclude that cyanobacterial or- offer more of a burden than benefit to waste-treatment
ganic carbon contribution is not important in these efficiency. However, the majority of waste-treatment
systems, but could prove to be important at sites that systems experience a net-decrease in BOD and toxic-
achieve lower BOD loadings following mill upgrades ity, even with the presence of large cyanobacterial
and process changes. communities. Rather, the magnitude of this net de-
The potential interactions between cyanobacteria crease is highly variable, and may be due to differ-
and the heterotrophic community could involve com- ences beetween sites in microbial community struc-
petition for both nutrients and organic substrates. In ture and dynamics.
systems undergoing fertilization, nutrient competition
would be less intense, but competition for organic
substrates between cyanobacteria and bacteria is a
334

Acknowledgements Hansman E. 1973. Chap. 23, 359–368. Pigment analysis. In: Stein
Janet R. (ed.), Handbook of Phycological Methods. Culture
Methods and Growth Measurements. Cambridge University
The authors thank the University of Toronto Pulp and Press, 448 pp.
Paper Industrial Research Consortium members Harris G.P., Haffner G.D. and Piccinin B.B. 1980. Physical vari-
Georgia-Pacific, Potlatch, Tembec and Tasman for ability of phytoplankton communities II. Primary productivity
providing the wastewater for our study. Financial as- by phytoplankton in a physically variable environment. Arch.
sistance was provided to R. Fulthorpe by the Univer- Hydrobiol. 88: 303–327.
Hausalo T. 1995. Analysis of wood and pulp carbohydrates by an-
sity of Toronto Pulp and Paper Industrial Research ion exchange chromatography with pulsed amperometric detec-
Consortium and the National Science and Engineer- tion. Proc. 8th International Symposium on Wood and Pulping
ing Research Council (NSERC). This research was Chemistry (ISWPC), Helsinki, Finland 3: 131–136.
also supported by a NSERC grant to C. Nalewajko. Jansson M. 1993. Uptake, exchange, and excretion of orthophos-
phate in phosphate-starved Scenedesmus quadricauda and
Pseudomonas K7. Limnol. Oceanogr. 38: 1162–1178.
Jarvinen R. 1997. Nitrogen in the effluent of the pulp and paper
References industry. Wat. Sci. Tech. 35: 139–145.
Khoja T.M. and Whitton B.A. 1971. Heterotrophic growth of blue-
Allen M.M. 1968. Simple conditions for the growth of unicellular green algae. Arch. Mikrobiol. 79: 280–282.
blue-green algae on plates. J. Phycol. 4: 1–4. Kirkwood A.E., Nalewajko C. and Fulthorpe R.R. 2001. The oc-
American Water Works Association 1995. Standard Methods for currence of cyanobacteria in pulp and paper waste-treatment
the Examination of Water and Wastewater. 19th edn., Denver, systems. Can. J. Microbiol. 47: 761–766.
Colorado, USA. Kuritz T. and Wolk C.P. 1995. Use of filamentous cyanobacteria
Bagchi S.N., Chauhan V.S. and Palod A. 1990. Heterotrophy and for biodegradation of organic pollutants. Appl. environ. Micro-
nitrate metabolism in a cyanobacterium Phormidium uncina- biol. 61: 234–238.
tum. Current Microbiol. 21: 53–58. Lean D.R.S. and Nalewajko C. 1979. Phosphorus turnover time
Beardall J. and Morris I. 1976. The concept of light intensity ad- and phosphorus demand in large and small lakes. Arch. Hydro-
aptation in marine phytoplankton: some experiments with biol. 13: 120–132.
Phaedoactylum tricornutum. Mar. Biol. 37: 377–387. Liss S.N., Bicho P.A. and Saddler J.N. 1997. Microbiology and
Bryant C.W., Barkley W.A., Garrett M.R. and Gardner D.F. 1997. biodegradation of resin acids in pulp mill effluents: a minire-
Biological nitrification of kraft wastewater. Wat. Sci. Tech. 35: view. Can. J. Microbiol. 75: 599–611.
147–153. Liukko S. and Poppius L.K. 1999. Characteristics of dissolved or-
Chapman D.J. 1973. Biliproteins and bile pigments. In: Carr N.G. ganic material in total chlorine free bleach plant and laboratory
and Whitton B. (eds), The Biology of Blue-Green Algae. Bot. effluents. Wat. Sci. Tech. 40: 249–258.
Monogr. 9. University of California Press, Berkely, USA, pp. Marquez F.J., Sasaki K., Kakizoo T., Nishio N. and Nagai S. 1993.
675. Growth characteristics of Spirulina platensis in mixotrophic
Currie D.J. and Kalff J. 1984. A comparison of the abilities of and heterotropic conditions. J. Ferment. Bioeng. 76: 408–410.
freshwater algae and bacteria to acquire and retain phosphorus. Narro M.L. 1987. Petroleum toxicity and the oxidation of aromatic
Limnol. Oceanogr. 29: 298–310. hydrocarbons. In: Fay P. and Baalen C.V. (eds), The Cyanobac-
Currie D.J. 1990. Large-scale variability and interactions among teria. Elsevier Science, pp. 493–511.
phytoplankton, bacterioplankton and phosphorus. Limnol. Narro M.L., Cerniglia C.E., Van Baalen C. and Gibson D.T. 1992.
Oceanogr. 35: 1437–1455. Metabolism of phenanthrene by the marine cyanobacterium
Falkowski P.G. 1984. Physiological responses of phytoplankton to Agmenellum quadruplicatum PR-6. Appl. environ. Microbiol.
natural light regimes. J. Plankton Res. 6: 295–307. 58: 1351–1359.
Fogg G.E., Stewart W.D.P., Fay P. and Walsby A.E. 1973. The Nikkila A., Paulsson M., Almgren K., Blanck H. and Kukkonen
Blue-Green Algae. Chap. 9. 161–179. Academic Press, London J.V.K. 2001. Atrazine uptake, elimination and bioconcentration
& New York, 459 pp. by periphyton communities and Daphnia magna: effects of dis-
Fulthorpe R.R. and Allen D.G. 1995. A comparison of organochlo- solved organic carbon. Environ. Toxicol. Chem. 20:
rine removal from bleached kraft pulp and paper-mill effluents 1003–1011.
by dehalogenating Pseudomonas, Anclyobacter and Methylo- Paerl H.W., Bebout M.S., Joye B. and Des Marais J. 1993. Micros-
bacterium strains. Appl. Microbiol. Biotechnol. 42: 782–789. cale characterization of dissolved organic matter production
Gapes D.J., Frost N.M., Clark T.A., Dare P.H., Hunter R.G. and and uptake in marine microbial mat communities. Limnol.
Slade A.H. 1999. Nitrogen fixation in the treatment of pulp and Oceanogr. 38: 1150–1161.
paper wastewaters. Wat. Sci. Tech. 40: 85–92. Pinckney J., Paerl H.W. and Fitzpatrick M. 1995. Impacts of sea-
Gauthier F., Neufeld J.D., Driscoll B.T. and Archibald F.S. 2000. sonality and nutrients on microbial mat community structure
Coliform bacteria and nitrogen fixation in pulp and paper mill and function. Mar. Ecol. Progr. Ser. 123: 207–216.
effluent treatment systems. Appl. environ. Microbiol. 66: 5155– Puhakka J.A., Maikinen P.M., Lundin M. and Ferguson J.F. 1994.
5160. Aerobic and anaerobic biotransformations and treatment of
chlorinated pulp bleach waste constituents. Wat. Sci. Tech. 29:
73–80.
335

Radwan S.S. and Al-Hasan R.H. 2000. Oil Pollution and cyano- Stewart W.D.P. 1964. The effect of available nitrate and ammoni-
bacteria. Chap. 11. In: Whitton B.A. and Potts M. (eds), The um-nitrogen on the growth of two nitrogen-fixing blue-green
Ecology of Cyanobacteria. Kluwer Academic Publishers, The algae. J. exp. Bot. 15: 138–145.
Netherlands, pp. 307–319. Twiss M.R., Granier L., Lafrance P. and Campbell P.G.C. 1999.
Reuter J.E., Loeb S.L. and Goldman C.R. 1986. Inorganic nitrogen Bioaccumulation of 2,2⬘,5,5⬘-tetrachlorobiphenyl and pyrene
uptake by epilithic periphyton in a nitrogen deficient lake. Lim- by picoplankton (Synechococcus leopoliensis, Cyanophyceae):
nol. Oceanogr. 31: 149–160. Influence of variable humic acid concentrations and pH. Envi-
Rippka R. 1972. Photoheterotrophy and chemoheterotrophy among ron. Toxicol. Chem. 18: 2063–2069.
unicellular blue-green algae. Arch. Mikrobiol. 87: 93–98. Vollenweider R.A. 1969. A manual on methods for measuring pri-
Sahu J. and Adhikary S.P. 1982. Heterotrophic growth and pigment mary productivity in aquatic environments. IBP Handbook No.
composition of four filamentous blue-green algae. Arch. Hy- 12. Blackwell Scientific, UK.
drobiol. Suppl. 63: 189–199. Wall D. and Briand F. 1979. Response of lake phytoplankton com-
Schmidt T.S.K., Simkins S. and Alexander M. 1985. Models for munities to in situ manipulations of light intensity and colour.
the kinetics of biodegradation of organic compounds not sup- J. Plankton Res. 1: 103–111.
porting growth. Appl. environ. Microbiol. 50: 323–331. Wetzel R.G. 1983. Limnology. 2nd edn. Sanders College Publish-
Shashirekha S., Uma L. and Subramanian G. 1997. Phenol degra- ing, New York, USA, pp. 767.
dation by the marine cyanobacterium Phormidium valderianum
BDU 30501. J. indust. Microbiol. Biotech. 19: 130–133.