Biocrude Production by Activated Sludge

using amended Pulp and Paper wastewater

as substrate

Kamal Lamichhane , Andro Mondala, Rafael Hernandez, Todd French, Magan Green,
Linda McFarland, William Holmes
Dave C. Swalm School of Chemical Engineering, Mississippi State University

Content
Objective
Introduction
Materials and methods
Results and
discussions
Conclusion


Objective
The objectives of this study were to:
1. Test pulp and paper waste water to grow
microorganisms capable of lipid
accumulation, and measure the lipid
accumulation and biodiesel yield
2. Compare the lipid and biodiesel yield
using pulp and paper wastewater with
synthetic wastewater



Wastewater Treatment Plant for Enhancement of lipid
yield
Screening/grit
removal
Aeration tank
Clarifie
r
Air
Air
Clarifie
r
Domestic/industrial
Wastewater
influents
Effluent
Oily
sludge to
lipid
extraction
steps
Belt press
Activated
sludge
Extruded
water
Clarifier
Lipid
accumulation
chamber
Carbon and
nutrient
sources
Recycle
Holding
tanks
Primary
sludge to
anaerobic
digestion or
lipid
extraction
Effluen
t
Waste
sludge
Pulp and Paper wastewater
Approximately 1551 billion
gallons of wastewater
produced annually in the
United States
1
For every ton of pulp
produced, 17,000 galloons
of industrial process water
required
2
Almost half of the total
waste is produced in the
southern region

Fig1: International Paper, Vicksburg, MS
1Effluent Limitations and Guidelines, Pretreatment Standards, and New Source Performance Standards: Pulp, Paper and
Paperboard Category; National Emission Standards for Hazardous Air Pollutants for Source Category; Pulp and Paper
Production; Proposed Rule, Dec. 17, 1993
2. http://chlorinefreeproducts.org/images/Article3.pdf
Pulp and Paper Wastewater:
characteristics and common
contaminants
Characteristics
High COD (500-1500 mg/L)
Dark brown coloration
Adsorbable organic halide
Toxic pollutants
Common Contaminants
Sodium carbonate
Sodium hydroxide
Sulfates
Chlorine dioxide
Calcium oxide

Manufacturing Process

Wood
Preparation
Cooking(NaOH
and Na
2
S)
Pulp
Washing
Pulp
Screening
Bleaching
Wood Preparation: removes bark
Cooking : most of the lignin removed from the
wood
Pulp washing: wash out the cooking chemicals
and lignin from wood fiber
Pulp screening: remove the uncooked fibers
Bleaching: two stage bleaching-
a) Pulp treated with NaOH in presence of
O
2
to break down the polymer
b) Pulp is treated with ClO
2
to remove the
remaining the lignin

Wastewater From Pulp and Paper
Mills
Wastewater Treatment
Process
Fig 2: Flow Scheme of general Pulp and Paper Wastewater Treatment Process*

*Ince, Bahar K., Cetecioglu, Zeynep., Ince, Orhan (2011).Pollution prevention in the pulp and
paper Industry. pp 231.

Activated Sludge Process
Biological method that uses
microorganisms to feed on
organic contaminants in
wastewater
The microorganisms form floc
and settle at the bottom
leaving clear liquid at the top
The top liquid is free of
organic materials and
suspended solids
*
*Expanding the Activated Sludge Process (2003). Retrieved September 23, 2012 from
http://www.nesc.wvu.edu/pdf/ww/publications/pipline/pl_sp03.pdf
Fig3: typical activated sludge process
http://www.enotes.com/topic/Activated_sludge
Oleaginous microorganisms
Activated Sludge contains a
mixed microbial community
Oleaginous microorganisms in
activated sludge can
accumulate a high percentage
of lipids
Microorganisms require water
with high organic content (high
C:N ratio) to grow
Pulp and paper wastewater
could be a suitable growth
media for microorganisms
specialized in biocrude
production
Fig 4: Cells of an Oleaginous yeast
*
*. Wynn, James P. Ratledge, Colin. (2005) Oils from Microorganisms pp 123.
Wastewater Initial Characteristics
Fig 5: Different types of pulp and
paper wastewater
Characteristi
cs
Pulp Wash
Mixed
Wastewater
Pond Effluent
Process
derived from
Wood pulping,
bleaching, and
washing.
Combined
wastewater
streams
from mill.
Effluent from
anaerobic
settling pond.
pH
2.25 6.26 8.59
COD (mg/L)
1478 1361 410
Ammonium
(mg/L)
3.95 5.90 2.83
Sulfate
(mg/L)
1400 840 400
Materials and Methods
Following fermentation experiment was
set up in the bioflo 310 fermenter
2.4 L of the wastewater
20% Activated sludge ( 600 ml)
60 g/L sugar
1.3 g/L Ammonium chloride
1.5 g/L of Potassium phosphate and
1g/L of sodium phosphate
The initial pH was set to 6.5 and agitation
was maintained at 300 rpm and changed
to maintain the DO at 60% saturation
Fig 5: Bioflo 310
Fermenter
Experimental Procedure
Activated
Sludge+
wastewater
Fermentation
Centrifuge
NH4+, COD, sugar
Freeze dry
Bligh and
Dyer+TurboVap
Transesterification
GCFID
Kinetics: Biomass growth
Mathematical modeling was done following the methods by Sattur
et al. (1991), Karanth et al. (1991) and Mondala et al. (2011)
The biomass growth was determined using the logistic model given
by Weiss and Ollis

max
max
max 0
max
max
max
max
max
max
slope with line straight a gives t vs ln of plot
ln 1 ln t
g, Reaggangin
biomass Maximum
rate growth specific Maximum
biomass lipid non of ion Concentrat X(t) where,
) (
1 ) (
|
|
.
|

\
|

|
|
.
|

\
|
+
|
|
.
|

\
|
=
=
=
=
|
|
.
|

\
|
=
X
X
X
X
X
X
X
X
X
X
t X
t X
dt
dX

Kinetics: Lipid Accumulation

Lipid accumulation was modeled using Luedking-Piret
equation

Vs. ( X X
0
) gives straight line
with slope m
Plotting
After linearization, the final values of the kinetic parameters were
estimated by non-linear regression using the Levenberg-Marquardt
method in Polymath 6.1
Po = Initial Lipid concentration
n= stationary phase constant
m= growth phase constant
Kinetics: Substrate
consumption
Substrate consumption was modeled by a Luedeking- Piret
equation

X K
dt
dP
Y
m
dt
dX
Y dt
dS
e
S P S X
+ + =
/ /
1
e
S P
K
Y
n
+ =
/
|
S P S X
Y
m
Y
/ /
1
+ = o
( )
O
O
O
X t X
t X
X
t
X
S t S
(

|
|
.
|

\
|
+ = ) (
) (
ln ) (
max
max
max
o

|
S
0
, X
0
= initial concentration of sugar, and non lipid biomass respectively

max
= Maximum specific growth rate
K
e
= Maintenance coefficient
, = constants used to predict residual sugar maintenance coefficient
Y
x
= biomass yield based on sugar consumed
Y
p
= Lipid yield based on sugar consumed
Results : Pulp Wash
0
50
100
150
200
250
300
350
400
0
10
20
30
40
50
60
0 24 48 72 96 120 144 168
A
m
m
o
n
i
u
m
(
m
g
/
L
)

B
i
o
m
a
s
s
,

L
i
p
i
d
,

G
l
u
c
o
s
e



Time(hr)
biomass (g/L)
glucose(g/L)
lipid (%w/w)
ammonium(mg/L)
Results : Pond Water
0
50
100
150
200
250
300
350
400
450
0
10
20
30
40
50
60
0 24 48 72 96 120 144 168
A
m
m
o
n
i
u
m

B
i
o
m
a
s
s
,

L
i
p
i
d
,

G
l
u
c
o
s
e

Time (hr)
biomass(g/L)
lipid(%w/w)
glucose(g/L)
ammonium (mg/L)
Results: Mixed wastewater
0
50
100
150
200
250
300
350
400
0
10
20
30
40
50
60
0 24 48 72 96 120 144 168
A
m
m
o
n
i
u
m

B
i
o
m
a
s
s
,

L
i
p
i
d
,

G
l
u
c
o
s
e

Time (hr)
biomass (g/L)
glucose (g/L)
lipid (%w/w)
ammonium (mg/L)
Result: Lipid and Fames
Wastewater
Biocrude
(%w/w)
Biodiesel
(%w/w)
Pulp Wash 16.04 6.22
Pond Water 29.59 7.45
Mixed wastewater 10.77 5.9
Fatty Acid Pulp Wash Pond Water Mixed Waste
Myristic (C14:0) % 1.16 0.38 1.03
Palmitic (C16:0) % 19.56 19.4 19.35
Palmitoleic (C16:1) % 3.42 3.44 3.9
Stearic (C18:0) % 11.4 11.03 11.37
Oleic (C18:1) % 44.94 50.26 44.82
Linoleic (C18:2) % 14.93 13.15 15.19
Linolenic (C18:3) % 2.41 1.83 1.93
Arachidic (C20:0) % 0.69 0.36 0.52
Lignoceric (C24:0) % 0 0.15 1.64
Table: Fatty Acid Profile
Kinetic Model Parameters
Biomass growth
X
o
7.7347
X
max
15.110

max
0.008837
R
2
0.7676
Lipid Accumulation
m 0.1352
n 0.0004697
R
2
0.8054
Sugar Consumption
1.5849
0.021948
R
2
0.9622
Lipid Accumulation
m 0.958
n 0.00304
R
2
0.80158
Biomass growth
X
o
8.67
X
max
17.05

max
0.003528
R
2
0.40043
Sugar Consumption
8.5732
0.0172246
R
2
0.90832
Lipid
Accumulation
m 0.0319
n 0.0005047
R
2
0.76337
Biomass growth
X
o
8.29879
X
max
19.2200

max
0.012329
R
2
0.82707
Sugar Consumption
1.8159
0.0185692
R
2
0.9598
Pulp Wash
Pond Water Mixed Waste
Remarks
1. All of the three wastewaters were suitable for
oleaginous microbial growth when amended with
sugar and other nutrients
2. Microorganisms growing in pond water accumulated
most lipid (29.59 % CDW) at day 7 compared to pulp
wash (11.69%) and mixed wastewater (8.19%)
3. The biodiesel yield was around 6-7 % W/W for all the
wastewater
4. Fatty acid profile was similar to those obtained from
domestic wastewater

Future Work
Optimization of fermentation conditions and
investigation of fed-batch or continuous modes.
Identification of possible toxic compounds and
determination of their fate in activated sludge
lipid accumulation cultures.
Correlate BOD and COD reduction with
biocrude production.
Utilization of pulp and paper mill solid wastes
and fiber as source of fermentable sugars.