596 of Scientific & Industrial Research

Journal J SCI IND RES VOL 72 SEP - OCT 2013

Vol. 72, Sept - Oct 2013, pp. 596-602

Bioprocess development for utilizing biodiesel industry generated crude glycerol for
production of poly-3-hydroxybutyrate

Mathew Jincy, Raveendran Sindhu, Ashok Pandey and Parameswaran Binod*

Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum 695 019, India

Received 19 March 2013; revised 13 May 2013; accepted 29 May 2013

This study presents Bacillus firmus NII 0830 for producing poly-3-hydroxybutyrate (PHB) using biodiesel industry
generated glycerol, a waste by-product, as sole carbon source under submerged fermentation and production process was
optimized using statistical experimental design. PHB accumulation was observed up to 1.60 g/l from 4.36 g/l of total bacterial
biomass at inoculum size of 3% v/v, incubation temperature 30°C, crude glycerol concentration 5% v/v, 250 RPM, incubation time
60 h and media pH 6.0 during initial statistical design. Further Response Surface design experiments yielded 61% of PHB from B.
firmus NII 0830. Glycerol has a low market price and it does not require any further refinement, makes present process more
economical. Further, utilization of low-grade waste glycerol solves its disposal problem, hence it is aptly an environmentally
friendly ‘waste to wealth’ bioprocess.

Keywords: Bacillus firmus, Crude glycerol, Polyhydroxybutyrate and Response surface methodology (RSM)

Introduction cost, product yield and product recovery method6. Raw

Poly-3-hydroxybutyrate (PHB) based polymers have
almost similar properties when compared with
polypropylene, a widely used petroleum-based synthetic
polymer. PHB is biodegradable and potentially useful for
food packaging films, biodegradable carriers for
medicines and insecticides, disposable cosmetic products
(razors and diapers), absorbable surgical devices and
starting compounds for chiral substances1. Microbial
synthesis of PHB takes place if a suitable carbon
substrate is available in excess and cellular growth is
limited by other nutrients like nitrogen or phosphate.
Ralstonia eutropha is the most widely used organism
for PHB production because it can accumulate large
amounts of PHB (up to 80% of dry cell weight) in a
simple medium, and its physiology and biochemistry
leading to PHB synthesis are best understood2. Main
limitation for industrial production of biopolymers is the
difficulty involved in synthesising from inexpensive
precursors and high cost of their recovery3,4. Biosynthetic
pathways and regulation helped in genetic and metabolic
engineering and have allowed construction of recombinant
microbes with improved production yields3,5. Factors
affecting PHB production cost include carbon substrate
*Author for correspondence
E-mail: binodkannur@niist.res.in
materials cost accounts for over 50% of the total
production cost and out of this carbon source accounts
for 70- 80% of the total raw material cost7. Thus cost of
raw materials plays a significant role in overall economics
of PHB production in pilot scale. Alcoholic distillery
effluent8, potato processing waste9, corn steep liquor10,
molasses11, milk whey12, crude glycerol (CG)13,14 and
agro-residues15 were used to replace the much expensive
carbon and nitrogen sources. Biodiesel manufacturing
processes create huge amounts of CG co-product waste
stream, which could be used as a promising carbon
source for microbial fermentation for PHB production.
Several reports are available for PHB production from
glycerol 16 . Depending on manufacturing process
employed, glycerol concentration varies between 62 and
90 w%17. PHB produced using CG exhibit properties
similar to those produced from glucose18. In the process
of conversion of glycerol to PHB, bacteria first convert
glycerol into dihydroxyacetone and through various
biochemical reactions it is finally converted into acetyl-
CoA, which is converted into PHB after enzyme catalysed
reactions (Fig. 1). Utilization of waste glycerol would
lower biopolymer production cost by utilizing un-purified
cheap carbon source concomitantly with harnessing
waste disposal.
 JINCY et al: BIOPROCESS USING CRUDE GLYCEROL FOR POLY-3-HYDROXYBUTYRATE PRODUCTION
Fig. 1—Biochemical pathway involved in bacterial produciton of PHB from glycerol
This study presents a fermentation strategy for PHB
production by Bacillus firmus NII 0830 using biodiesel
industry generated CG as sole carbon source as well as
statistical optimizations of nutritional and environmental
conditions for growth as well as PHB production.
Experimental Section
Chemicals and Reagents
CG obtained as a by-product from biodiesel industry
(Yantra Fintech India Pvt Ltd, Chennai, India), had
following components (v/v): glycerol, 82; moisture, 3.0;
free fatty acid (FFA), 4.5; ash, 6.0; ester, 1.0; methanol,
0.1; and other organic matter, 8.0%. Crotonic acid and
PHB standards were obtained from Sigma-Aldrich, India.
All other chemicals were of reagent grade procured from
local vendors.
Microorganisms and Growth Conditions
Microorganisms, B. firmus NII 0830, were
maintained on nutrient agar slants. Inoculums, prepared
in tubes containing Luria-Bertani (LB) media, were
incubated at 30°C for 16 h on a rotary shaker at 200
rpm. Seed culture (2% v/v) was transferred to
fermentation media (100 ml) as and when required.
597
Fermentative Production of PHB
PHB production was carried out in 250 ml
Erlenmeyer flask containing 50 ml culture media, which
had following composition: CG, 10; (NH4) 2SO 4 , 4;
K2HPO4, 2; MgSO4.7H2O, 1.2; and citric acid, 1.7 g/l.
Composition of trace element solution (10 ml/l) was:
FeSO4.7H2O, 10; ZnSO4.7H2O, 2.25; CuSO4.5H2O, 1;
MnSO4 .5H2O, 0.5; CaCl2.2H2O, 0.2; Na2B4O7.7H2O,
0.23; and (NH4)8Mo7O24, 0.1 g/l; and 35% HCl, 10 ml/l.
Media were inoculated with 2% inoculum containing
5x106 CFU/ml, incubated at 30°C up to 72 h, centrifuged
and biomass pellets were separated and lyophilized.
PHB Assay
PHB assay19 was carried out by converting PHB
into crotonic acid by acid hydrolysis and estimating
crotonic acid by High Perfor mance Liquid
Chromatography (HPLC). Lyophilized pellet (0.5 g) was
digested in conc. sulphuric acid (1 ml) at 90°C for 30
min, cooled and then mixed with 0.014 N H2SO4 (4 ml).
Sample was diluted 50 times using 0.014 N H2SO4 and
filtered using 0.2 µm filter (Ultipore N66 Nylon 6, 6
membranes). Sample (10-50 µl) was injected in HPLC
fitted with Aminex HPX-87 H ion exclusion organic acid
 598 J SCI IND RES VOL 72 SEP - OCT 2013

Table 1—Experimental design

Run Inoculum Incubation Medium Incubation Glycerol

size temp. pH RPM time conc. v/v Biomass PHB
% °C h % g/l g/l
1 3 35 8.00 150 36 5 1.42 0.19
2 1 30 8.00 250 60 5 5.04 0.44
3 3 30 6.00 250 36 1 4.13 0.40
4 3 35 8.00 250 60 5 2.53 0.28
5 1 35 6.00 250 36 1 1.40 0.53
6 3 35 6.00 250 36 5 3.20 0.48
7 3 30 8.00 150 36 1 2.23 0.77
8 1 30 6.00 250 36 5 4.36 0.57
9 3 30 6.00 150 60 5 3.95 0.52
10 3 30 6.00 150 36 5 2.50 0.70
11 1 35 8.00 250 60 1 2.65 0.37
12 3 35 8.00 250 36 1 2.13 0.28
13 1 35 6.00 150 36 5 1.38 0.14
14 3 35 6.00 150 60 5 1.99 0.20
15 1 30 8.00 150 36 5 2.28 0.43
16 1 35 8.00 150 36 1 1.02 0.19
17 3 30 6.00 250 60 5 4.36 1.60
18 1 35 6.00 150 60 1 1.35 0.28
19 1 35 8.00 150 60 5 1.25 0.26
20 3 35 6.00 150 36 1 1.32 0.26
21 1 30 6.00 250 60 1 1.28 0.19
22 3 30 6.00 150 60 1 2.98 0.73
23 1 30 6.00 150 60 5 3.70 1.12
24 1 35 8.00 250 36 1 1.28 0.59
25 1 35 8.00 250 36 5 2.06 0.51
26 3 30 8.00 250 60 1 2.04 0.69
27 3 35 6.00 250 60 1 2.28 0.47
28 3 30 8.00 150 60 5 2.38 1.12
29 1 30 8.00 150 60 1 2.86 0.96
30 1 30 8.00 250 36 1 2.55 1.49

analysis column (300 mm x 7.8 mm) preceded by an
organic acid guard column (Biorad). Samples were eluted
with 0.014 N H 2 SO 4 (flow rate, 0.6 ml/min) and
absorbance was taken at 235 nm, with crotonic acid as
standard using a Diode Array Detector (Shimadzu,
Japan).
Statistical Optimization for PHB Production
For statistical optimization, response surface
methodology (RSM) was designed by Design Expert
Version 7.1.6 (Stat-Ease, Inc., Minneapolis) using
Minimum Run Equi-replicated Resolution V Design
method. All variables were denoted as numerical factors
and investigated at two widely spaced intervals
designated as -1 (low level) and +1 (high level).
Parameters (7) were chosen for experimental design
(Table 1) and their two levels were: age of inoculums, 6
& 16 h; inoculums size, 1 & 3% v/v; incubation temp.,
30 & 35oC; pH of medium, 6.0 & 8.0; RPM, 150 & 250;
incubation time, 36 & 60 h; and CG conc., 1 & 5% v/v.
Effects of individual parameters on PHB and biomass
production was calculated as E = (SM+ - SM-)/N, where
E is effect of parameter, M+ and M– are responses (PHB
and biomass) of trials at which parameter was at its
higher and lower levels respectively, and N was total
number of trials. From effect estimate plot, developed
using Microsoft Excel 2007, parameters showing
maximum effect on PHB production were selected for
Box-Behnken design. Parameters were studied at three
different levels in a 13 runs experimental RSM design
(Table 2).
Results and Discussion
Glycerol content in CG sample was found to be 82%.
Generally, glycerol purity from biodiesel generation
process is reported between 80-85%17,20. Even though
CG contains methanol, salts as well as other components,
B. firmus NII 0830 can grow and produce PHB utilizing
 JINCY et al: BIOPROCESS USING CRUDE GLYCEROL FOR POLY-3-HYDROXYBUTYRATE PRODUCTION 599

Table 2—RSM design

Run order Temp. Glycerol Biomass PHB PHB
°C % g/l g/l %
1 37 2 0.689 0.348 50.452
2 30 3.5 0.886 0.478 53.976
3 30 3.5 0.878 0.497 56.591
4 25 5 0.414 0.216 52.056
5 25 2 0.991 0.605 60.981
6 37 3.5 0.721 0.355 49.160
7 30 5.5 0.424 0.214 50.518
8 30 3.5 0.845 0.468 55.298
9 37 5 0.446 0.255 57.129
10 30 3.5 0.898 0.468 52.109
11 30 3.5 0.855 0.423 49.551
12 30 1.5 0.922 0.544 59.006
13 25 3.5 0.955 0.548 57.363
Table 3—Statistical analysis of model

Source Sum of squares Degree of freedom Mean square F value Prob>F

Model
A-Inoculum Age 18.80 8 2.35 3.16 0.0164
B-Inoculum size 0.013 1 0.013 0.018 0.8958
C-Incubation temp. 0.30 1 0.30 0.41 0.5307
D-Medium pH 11.22 1 11.22 15.08 0.009
G-Glycerol conc. 0.19 1 0.19 0.25 0.6226
Residual 3.24 11 3.24 1.36 0.0492
Corr. 15.62 21 0.74
Total 34.42 29
CV – 11.2 R2 – 0.858

Fig. 2—Effect estimate on A) PHB and B) biomass production

CG as sole carbon source without any pretreatment.
Among different strains screened for PHB production14
using CG, B. firmus NII 0830 was found to be the best
producer of PHB. Biomass as well as PHB production
as a function of 7 parameters was evaluated by using a
Minimum Run Equi-replicated Resolution V Design
method. The model was analyzed using a Design Expert
(Table 3). F value (3.16) implies that model was
significant. There was only a 1.64% chance that a
“Model F-Value” this large could occur due to noise.
Values of “Prob > F” (< 0.0164) indicated that model
terms were significant. In this case, incubation
temperature and CG conc. were significant model terms.
Final equation in terms of coded factors was
 600 J SCI IND RES VOL 72 SEP - OCT 2013
a)
Fig. 3—Interaction plot for glycerol concentration and incubation temperature on: a) biomass production; and b) PHB production
Biomass = 2.46 +0.022 * A +0.10 * B -0.63 * C - 0.083
* D + 0.33 * G -0.21 *A*B -0.15 * C * G -0.073 *D*G
PHB = +5.76735 +0.087393 * A + 0.56105 * B -0.15887
* C + 0.026475 * D+1.42127 * G -0.041486 * A* B -
0.030726 * C * G - 0.036501*D*G
Effect estimate (Fig. 2) on biomass production and
PHB showed that inoculum age and medium pH had a
positive effect and incubation temperature and CG conc.
had a negative effect on PHB production. On biomass
production, CG conc. had a positive effect and incubation
temperature had a negative impact. To find an optimum
medium for both biomass as well as PHB production
these two variables were selected for further optimization
under RSM using a three level variable (Table 2). From
RSM experiment, maximum biomass formation (0.991
g/l) and PHB production (0.60 g/l) was recorded at 25°C
with 2% (v/v) CG conc. Under these condition, PHB
production was 61% of biomass.
Under interaction plot on biomass production for CG
conc. and incubation temperature (Fig. 3a), biomass
production was low at middle and high levels of incubation
temperature. At low levels of incubation temperature and
CG conc., biomass production was high. Maximum
biomass production was observed at incubation
temperature 25-29°C. With an increase in temperature
above 29°C, there was a decline in biomass production.
Maximum values of cell dry weight, PHB yield and
productivities are reported21 at 30°C in B. mycoides RLJ
B -017. A contrary observation is reported22 for PHB
production using B. sphaericus, where maximum PHB
as well as biomass was produced at 30-35°C. Low level
of CG conc. (1.5-2.5%) showed maximum biomass
b
production. With an increase in CG conc. above 2.5%,
there was a decrease in biomass production. An identical
observation is reported18 for PHB production by C.
necator using NaCl contaminated CG, where PHB yield
coefficient was reduced from 0.37 g/g to 0.14 g/g during
time course of accumulation due to osmoregulation.
Under interaction plot for CG conc. and incubation
tempertaure on PHB production (Fig. 3b), at low levels
of incubation temperature and CG conc., PHB production
was high. Maximum PHB production was observed at
incubation temperature 25-29°C. With an increase in
temperature above 29°C, there was a decline in PHB
production. An identical observation is reported21 in B.
mycoides RLJ B -017. Low levels of CG conc. showed
maximum PHB production. Maximum PHB production
was observed at CG conc. Of 1.5-2.75%. With an
increase in CG conc. above 2.75%, there was a decrease
in PHB production. Decrease in PHB productivity at
higher CG conc. may be due to inhibition by other
components in CG. Ability of organism to grow and
accumulate PHB appears to be carbon specific21. Higher
CG conc. supported growth but were moderately used
for PHB accumulation.
The most favorable conditions for PHB as well as
biomass production were incubation temperature at 25°C
and CG conc. of 2% (v/v). Decrease in PHB
accumulation after cessation of cell growth is due to
intracellular consumption of PHB as energy and carbon
source. A number of Bacillus species accumulate PHB
from 4-66.7% dry cell weight. Carbon source provides
three functions within Bacillus species: i) a carbon source
for biomass synthesis; ii) an energy source for
biosynthesis and cell maintenance; and iii) a carbon source
 JINCY et al: BIOPROCESS USING CRUDE GLYCEROL FOR POLY-3-HYDROXYBUTYRATE PRODUCTION 601

Table 4—ANOVA for the model for PHB

Source DF Seq SS Adj SS Adj MS F P

Regression 5 0.189937 0.189937 0.037987 25.58 0.000
Linear 2 0.142484 0.142484 0.071242 47.97 0.000
Square 2 0.025593 0.025593 0.012797 8.62 0.013
Interaction 1 0.021860 0.021860 0.021860 14.72 0.006
Residual Error 7 0.010395 0.010395 0.001485
Lack-of-Fit 3 0.007476 0.007476 0.002492 3.41 0.133
Pure Error 4 0.002919 0.002919 0.000730
Total 12 0.200332

for PHB polymerization21 . Under ANOVA for RSM
model for PHB production (Table 4), values of probability
> F less than 0.05 indicated that model terms were
significant. R2- value was 93.65%. Model equation for
biomass and PHB in terms of coded factors was
Biomass = - 5.988 + 0.574 *A – 0.556 *B – 0.011A2 –
0.057B2 + 0.0277*A*B
PHB = -1.179 + 0.223*A – 0.635*B – 0.005 A2 – 0.026
B2 + 0.024*A*B
Currently PHB is produced in pilot scale from gram
negative bacteria (Cupriavidus necator, Alcaligenes
latus and recombinant E. coli)11, which has a drawback
that PHB isolated from these organisms contains outer
lipopolysaccharide (LPS) as well as endotoxins that will
copurify with PHB.
PHB production from glycerol has been investigated
in only a few studies23-27. Mothes et al18 used CG from
different biodiesel manufacturers as a substrate for PHB
production. However, significant decrease in PHB
productivity and product yields were recorded when
NaCl-contaminated crude glycerol was used.
Mohammad et al28 found a newly isolated bacterium,
Zobellella denitrificans MW1, able to produce large
amounts of PHB from glycerol, with enhanced growth
and polymer productivity in the presence of NaCl. Studies
using recombinant E. coli JM 109 harbouring genomic
DNA fragment from Streptomyces aureofaciens NRRL
2209 showed 60% (w/w) PHB content in bacterial cell
using a basal medium along with yeast extract, peptone
and glycerol29. A higher PHB content (76% w/w)) was
achieved by an unidentified osmophilic organism in a 42-
l fed-batch fermentation using glycerol liquor phase with
yeast extract and peptone25 . CG samples of different
manufacturers differ mainly in their contamination with
salts (NaCl or K2SO4), methanol, fatty acids and the
pH, due to the applied process. However, all these
feedstocks contain glycerol as a main component with
very low amounts of fatty acid soaps and residual fatty
acid methyl esters. Dobroth et al30 found that when mixed
microbial consortia was used for PHA production from
CG, methanol in CG was transformed to PHB. This study
also shows that B. firmus NII 0830 can utilize glycerol
contaminated with methanol (0.1% v/v) for PHB
production. According to present results, CG, is well
suitable as a competitive feedstock for biotechnological
production of PHB.
Conclusions
A novel strategy was developed utilizing biodiesel
industry generated CG as a feedstock for PHB production
without any pretreatment. B. firmus NII 0830 can grow
and produce PHB in CG contaminated with salts, fatty
acids and methanol. The most favorable conditions for
PHB as well as biomass production were incubation
temperature at 25°C and CG conc. of 2% (v/v), and at
this condition bacterial biomass could be able to
accumulate 61% PHB. This study shows that biodiesel
industry generated CG contaminated with ash, esters and
methanol is well suitable as a competitive feedstock for
biotechnological production of PHB. The major
advantage of developed process is utilization of by-
product from biodiesel industry without any
preprocessing.
Acknowledgements
Authors thank Department of Biotechnology, Govt
of India and Council of Scientific and Industrial Research
(CSIR), New Delhi for financial support.
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