Indian J Microbiol

DOI 10.1007/s12088-014-0509-1

REVIEW ARTICLE

Biodiesel Industry Waste: A Potential Source of Bioenergy

and Biopolymers
Prasun Kumar Sanjeet Mehariya
Subhasree Ray Anjali Mishra Vipin Chandra Kalia

Received: 14 December 2014 / Accepted: 22 December 2014

Association of Microbiologists of India 2014

Abstract Biodiesel has been recognized as a good source

of energy and its production is increasing rapidly. During
biodiesel production, effluent containing 7075 % glycerol
is generated, which accounts for 10 % of the total produce.
This has led to a scenario where the price of glycerol has
declined dramatically and its disposal is becoming uneconomical. Recent efforts to extend the biotechnological
applications of glycerol for producing hydrogen and
polyhydroxyalkanoates have been quite encouraging.
Bacillus spp. are among those few organisms, which have
the potential to metabolize glycerol to produce energy and
biopolymers. Bacillus may thus be recognized as the
Power-Horse of the future.
Keywords Bacillus
Biowaste
Dark fermentation

Hydrogen
PHA copolymers
Waste management

Introduction
Energy demand worldwide is increasing rapidly whereas
our abilities to produce it are not matching. It is resulting in
a scenario where declining fossil fuel reserves and rise in
environmental pollution are threatening the sustenance of
human beings. There is a growing awareness to look for
future fuels, which should be non-polluting, energy
P. Kumar (&)
S. Mehariya
S. Ray
A. Mishra
V. C. Kalia
Microbial Biotechnology and Genomics, CSIR-Institute of
Genomics and Integrative Biology (IGIB), Delhi University
Campus, Mall Road, Delhi 110007, India
e-mail: prasun.mcr@gmail.com; prasun.kumar@igib.in
P. Kumar
Department of Biotechnology, University of Pune, Pune 411007,
India

efficient, easy to transform into other forms and economical as well [1, 2]. Bioethanol and Biomethane have been
long recognized as potential energy sources, which can be
produced from wastes of biological origin. The most recent
entry into this category of clean and energy efficient fuel
has been the production of biodiesel. Biodiesel has already
achieved the distinction of being produced commercially.
Its production in the US and EU has reached a level of 6.9
million tonnes per year [3]. Incidentally, for every 100 t of
biodiesel produced, there is a concomitant release of 10 t
effluent containing 7075 % glycerol. The other contaminants in the effluent are methanol, soap, oils, salts, solid
organic materials etc. [4, 5]. With an unprecedented
demand for fuels, the production of biodiesel is soaring to
greater heights. The fallout of this industrial scale production of biodiesel has been the rapid decline in the
market prices of glycerol. It has touched such a low price
range that even its disposal is becoming uneconomical. It is
not leaving us with many options. This has lead to a
forthcoming problem of waste management and forced
researchers to look for bio-products which can add value to
the use of glycerol as feed [4, 6].
Fermentation of glycerol by Anaerobiospirillum has
been shown to produce succinic acid [7], citric acid by
Yarrowia lipolytica [8], 1,3-propanediol, acetic acid and
butyric acid by Clostridium butyricum [9], and 3-hydroxypropionic acid by Klebsiella pneumoniae [10, 11].
Whereas, Escherichia coli was able to metabolize it to a
host of bio-products such as ethanol, succinate, acetate,
lactate, high value omega-3 polyunsaturated fatty acids
[12] and hydrogen (H2) [13, 14]. In spite of a vast range of
potential applications of glycerol to get metabolized into a
wide range of products [15], the search for something more
substantial is still going on. Since, the demand for energy is
unlikely to go down, the best option will be to focus on

123

Indian J Microbiol

energy generating processes. With the identification of H2

as the future-fuel, biological H2 production (BHP) has
emerged as the best system of exploitation.

Biological Hydrogen Production

A large number of microbes have been reported with
abilities to metabolize a range of substrates of biological
origin into H2 [2, 1618]. Extensive amount of research
efforts have already been done to find out industrially
robust organism(s), which can metabolize various organic
matters, withstand adverse physiological conditions,
hydrolyse biowastes and transform the intermediates into
H2 and other bioactive molecules such as biopolymers etc.
[2, 16, 17]. Citrobacter, Clostridium, Enterobacter, Klebsiella, and Thermotoga are among those few organisms
which have been widely studied for their BHP abilities [17,
19]. More recent scrutiny of the large number of studies
done on BHP have made very interesting revelations: (1)
dark-fermentative process operative in batch culture can
lead to a H2 yield in the range of 0.163.8 mol/mol hexose
sugar, (2) photo-fermentative routes functioning in photosynthetic organisms can help in achieving a target of
0.237.2 mol H2/mol hexose sugar, (3) metabolism of
biowastes by dark-fermentative followed by photo-fermentative routes can allow H2 yields in the range of
0.68.3 mol H2/mol hexose sugar [2, 1719]. This
sequential fermentation route can allow us to surpass the
theoretical H2 yields of 4 mol/mol hexose sugar [2, 17, 20].
Here, we have presented the status of BHP from glycerol
and the organism(s) which can be exploited as H2-producer. Since BHP is still struggling to meet the challenges
of being rated as economically viable for commercialization, it becomes necessary to club this process with others
which can add value. Hence, in the next section we have
dealt with the use of glycerol as feed for producing
biopolymers.

Biotransformation of Glycerol to Hydrogen

Batch Culture
The biotransformation of glycerol as feed for H2 production
has been demonstrated during the last decade [3, 21].
Glycerol has emerged as a better and cheaper source of
carbon in comparison to the more conventional glucose. The
primary reason is its being more reduced than glucose, since
its metabolism generates more NAD(P)H, which can be
expected to result in higher H2 yields [3]. With pure glycerol
(PG) as feed, H2 yields (mol/mol) have been found to vary
with the type of organism used as H2-producer (Table 1): (1)

123

2.2 with Bacillus when 2 % glycerol was used as feed [1], (2)
1.11 with Clostridium at 1 % w/v feed [22], (3) 0.94 with
Citrobacter at 2 % w/v feed [23], (4) 0.891.05 with
Enterobacter at 25 % w/v feed [2327], (5) 0.94 with
E. coli at 1 % w/v feed [28], (6) 0.882.16 with Halanaerobium at 0.22 % w/v of feed [21, 29], (7) 0.340.61 with
Klebsiella spp. at 0.015 % w/v of feed [30], (8) 2.122.86
with Thermotoga maritima at 29210 mmol of feed and
0.82.75 with Thermotoga neapolitana at 0.251 % w/v of
feed [3, 23, 31]. It clearly indicates that these bacteria can
tolerate up to 2 % w/v of glycerol as feed and may exceptionally tolerate higher concentrations (Table 1).
In contrast to PG as feed, efforts have been also made to
transform crude glycerol (CG) to H2 (Table 1): (1) 0.74
with Clostridium at 1.64 % w/v feed [22], (2) 0.121.12
with Enterobacter at 0.173.1 % w/v feed [25, 32], (3)
0.140.34 with Klebsiella spp. at 1.67 % w/v feed [22, 33,
34], (4) 2.92 with Thermotoga at 0.3 % w/v of feed [31].
These studies provide necessary inputs for exploiting CG
as feed for these bacteria. It becomes all the more significant in view of the fact that CG contains a few contaminants which may prove inhibitory to bioprocesses [24, 35
37]. However, it has been shown that CG is as good as PG
since the trace amounts of contaminants was helpful in
improving bacterial growth and H2 production [26, 38]. In
fact, the efficiency to utilize CG has been revealed to be
higher when bacteria acted syntrophically to metabolize
contaminants such as methanol and soap present in the CG
[39]. In spite of these advantages, utilization of CG on a
large scale is still facing some hurdles.
Continuous Culture
For large scale production of H2, it is desirable to run the
biotransformation process in a continuous culture mode
(Table 2). Compared to batch culture studies listed above,
a limited quantity of effort has been made to demonstrate
H2 production in continuous culture [18, 22, 27, 40, 41]. A
few reports of continuous culture with Clostridium sp. have
shown that 0.51.05 mol H2/mol PG could be produced,
whereas with CG 0.77 mol H2/mol feed were produced at
1 % PG [3, 22, 27, 42]. In contrast to 1 % w/v of feed used
with Clostridium, 2 % w/v glycerol could be transformed
into 0.89 mol H2/mol feed. On the other hand, T. maritima
appears to be a high producer of H2, as it resulted in a net
yield of 2.41 mol/mol glycerol consumed. However, the
process may turn out to be uneconomical because the feed
concentration is very low0.25 % v/v. It thus requires a
much larger reactor volume to transform glycerol to H2
concentrations [3, 22, 27, 42].
Strict anaerobes such as Clostridium spp. shows product
inhibition. On the other hand, uninhibited growth of facultative anaerobe Enterobacter even at 100 % H2 permits

Indian J Microbiol
Table 1 Dark fermentative
hydrogen production from
glycerol: Batch culture

Organism

Glycerol
(%, w/w)

H2 Yield
(mol/mol)

References

Pure glycerol
Bacillus coagulans IIT-BT S1

2.2

[1]

Citrobacter freundii H3

0.94

[23]

Clostridium pasteurianum CH

1.11

[22]

Enterobacter aerogenes

0.89

[27]

E. aerogenes HU-101

1.05

[25]

E. aerogenes HU-101

10

0.89

[25]

E. aerogenes NRRL B407

10

116.4a

[24]

E. aerogenes ATCC35029

2.1

0.95

[26]

Enterobacter sp. H2

0.95

[23]

Escherichia coli MG165

Halanaerobium saccharolyticum sub. Senegalensis

1
0.2

0.94
1.21b

[28]
[21]

0.88b

H. saccharolyticum sub. Saccharolyticum

0.2

2.16b

Klebsiella pneumoniae GT1

0.01

0.619b

[30]

Thermotoga maritima DSM 3109

164a

2.84

[4]

210a

2.12

29a

2.86

Thermotoga neapolitana DSM 4359

198
T. neapolitana DSM 4359

[29]

2.45

0.25

2.65

[3]

0.3

1.04b

[31]

0.80b

C. pasteurianum CH4

1.64

0.74

E. aerogenes HU-101

0.17

1.12

[25]

2.5

0.71

[25]

E. aerogenes KKU-S1
E. aerogenes ATCC35029

3.1
2.1

0.12
0.95

[32]
[26]

K. pneumoniae DSM2026

2.0

0.53

[33]

Waste glycerol

a
b

mmol/L
Consumed basis

[22]

Klebsiella sp. HE1

1.6

0.14

[22]

Klebsiella sp. HE1

0.345

[34]

0.152

0.3

2.92

T. neapolitana DSM 4359

[31]

Table 2 Dark fermentative hydrogen production from glycerol: Continuous culture

Organism

Glycerol
(%, w/v)

Conditions

H2 yield
(mol/
mol)

References

Clostridium pasteurianum LMG 3285

pH 6.0; HRTa 12 h

1.05

[42]

C. pasteurianum CH4

pH 7.5; 35 C; HRT 12 h

0.50

[22]

C. pasteurianum CH4

pH 7.5; 35 C; HRT 12 h

0.77

[22]

Enterobacter aerogenes

pH 7.0; 37 C; 50 rpm; MYG medium

0.89

[27]

Thermotoga maritima DSM 3109

0.25

M-3 medium without HEPES YEb 2 g/L 300 rpm pH 7.0, 80 C

2.41c

[4]

Hydraulic retention time

Yeast extract
Consumed basis

123

Indian J Microbiol

continuous production [25, 43]. Thus, use of facultative

anaerobes such as Enterobacter, Citrobacter and Bacillus
etc. or a mixed culture of strict and facultative anaerobes
would be helpful in enhancing the substrate conversion
efficiency [1618, 41].
Although, Bacillus species have been shown to evolve H2
from pure sugars and biowastes [16, 18, 19, 40], however, the
role of glycerol as feed has been confined thus far only to
Bacillus coagulans [1]. In view of Bacillus being recognized
as a potential robust candidate for H2 production in future [2,
18], it is desirable to explore the abilities of other Bacillus
spp. as H2 producers from glycerol under dark fermentative
conditions. Bacillus spp. alone or in combinations have been
recently demonstrated to be effective as producer of hydrolytic enzymes, H2 and polyhydroxyalkanoates (PHAs) [16,
40, 4447]. Bacillus spp. have a unique ability of producing
the enzymeacyl-homoserine lactonelactonase, which
enable them to quench the quorum sensing system of competing bacteria within a community and help them to survive
[18, 48]. Since the fermentation process involves the active
participation of microbes, a comparison of H2 producers for
their H2 metabolic activities and other unique characteristics
has revealed that Bacillus can be rated as a strong contender,

especially for generating energy in the form of H2 from

biological waste materials [18].
Biotransformation of Glycerol
to Polyhydroxyalkanoates
Environmentalists are always worried about the accumulation of non-biodegradable plastics in the environment.
Plastics are being viewed as a drain on the fossil fuel
reserves, which are used as raw material for their production. Bioplastics (PHAs) have emerged as a promising
alternative to synthetic plastics [18, 49, 50]. Although
microbes are well known for their biotechnological applications, they are also known to accumulate biopolymers
(PHA) as food reserves [5153]. Production of PHA is
influenced by the type of feed (pure substrates as well as
biowastes) and the organism used [44]. Polyhydroxybutyrate, a form of PHA generally stored within the bacteria
during environmental stress is not found fit for commercial
production because of the high price of crude material,
brittleness, poor strength and the fact that its recovery
process is highly uneconomical. In order to circumvent
these limitations, production of PHA co-polymers by

Table 3 Abilities of microbes to convert glycerol to polyhydroxyalkanoate

Organisms

Feed
(%, w/v)

DCMa
(g/L)

Polyhydroxyalkanoate (PHA)
Content

Yield

References
c

Pure glycerol
Cupriavidus necator DMS 545

82.5

51.0

62.0

[49]

Methylobacterium rhodesienum MB 126

21.0

10.5

50.0

[49]

Escherichia coli K24KP

4.7

1.4

30.0

[58]

Bacillus megaterium

7.7

4.8

62.4

[49]

B. megaterium BBST4

5.7

3.4

60.0

[53]

B. megaterium BBST4

7.8

2.4

31.0

B. megaterium OU303A

24.8

15.5

62.4

[51]

B. licheniformis PHA007

10.4

6.6

68.8

[63]

B. cereus PHA037

3.3

2.0

60.7

[63]

B. thuringiensis R1

6.1

3.9

64.1

[54]

Bacillus sp. PHA023

7.3

4.4

60.6

[63]

Bacillus sp. 88D

2.5

1.5

60.4

[52]

Bacillus sp. CL1

0.27

2.7

1.4

52.1

[62]

Crude glycerol
Ralstonia eutropha ATCC17699

37.0

20.2

55.0

[56]

E. coli CT1061

38.0

19.0

51.0

[61]

Pseudomonas oleovorans NRRL B-14682

2.9

1.1

38.0

[60]

Bacillus sphaericus NII 0838

20.0

6.3

31.0

[64]
[59]

P. oleovorans NRRL B-14682

1.4

0.4

27.0

Pseudomonas corrugata 388

1.7

0.7

40.0

Dry cell mass

Polyhydroxyalkanoate in g/L

PHA produced (% of DCM)

123

Indian J Microbiol

employing biowastes as feed have been advocated. Use of

cheap raw materials such as biological wastes is anticipated
to help in reducing around 45 % of the total production
cost [50]. Due to its reduced physiological state, glycerol
has been reported as suitable substrate for PHA production
compared to pure sugars [54]. Most of the studies have
demonstrated the ability of various organisms to transform
glycerol into PHA. In most widely explored Gram negative
organisms, the PHA yield was in the range of 2762 % (w/
w), Cupriavidus necator being the best amongst all [55,
56]. Pseudomonas spp. are well known for their PHA
producing abilities [50, 57], which ranges from 27 to 40 %
w/w with glycerol as feed [5860]. Methylobacterium
rhodesienum and E. coli have been shown to produce
3051 % w/w of PHA on glycerol [61].
Remarkably, Bacillus spp. have been found to be efficient producers of PHA [18, 50, 62, 63]. With glycerol as
feed, Bacillus megaterium has been able to utilize 25 %
feed to produce 3162 % w/w PHA [49, 5153]. Bacillus
thuringiensis has been reported as the highest PHA producer with a capacity of 64.1 % (w/w) at 1 % PG (Table 3)
[54]. In contrast to many studies done on PG, a limited
effort has been made to metabolize CG. Bacillus sphaericus has been able to produce 31 % (w/w) PHA on 2 %
glycerol as feed [64]. However, the role of glycerol as feed
has been confined thus far only to B. coagulans [1].
Although, Gram negative bacteria such as Ralstonia and
Pseudomonas can produce only homopolymer, mcl-PHA
and its copolymers on glycerol as feed. However, Bacillus
spp. is the only organism that can grow well on glycerol (a
structurally non-related C-source) as feed and valorize it by
producing both homopolymer and copolymers of PHA [51,
52, 63].

Future Perspective
Among a large number of microbes, Bacillus is the only
genus among dark-fermentative microbes, which has been
shown to evolve H2 and PHA from pure sugars and
biowastes [16, 1820, 46]. As glycerol has been independently shown to have the potential of being transformed
into H2 and PHAs (Tables 1, 3), however, Bacillus is the
only organism which can produce both the bio-products
from this feed. It thus may be interesting to go for
sequential production, where the effluent from H2 production stage may be used as feed for PHA production.
Bacillus and its associated cultures are expected to fulfill
the demand of energy and minimize waste management
problems. Moreover, Bacillus has been well recognized as
industrial work-horse and as a GRAS organism, it will be
no surprise if Bacillus can be recognized as the PowerHorse for Future.

Acknowledgments The authors wish to thank the Director of CSIRInstitute of Genomics and Integrative Biology (IGIB), Delhi, CSIRWUM (ESC0108) Government of India for providing necessary funds
and facilities. P.K. is thankful to CSIR for granting Senior Research
Fellowship.

References
1. Kotay SM, Das D (2007) Microbial hydrogen production with
Bacillus coagulans IIT-BT S1 isolated from anaerobic sewage
sludge. Bioresour Technol 98:11831190. doi:10.1016/j.biortech.
2006.05.009
2. Kalia VC, Purohit HJ (2008) Microbial diversity and genomics in
aid of bioenergy. J Ind Microbiol Biotechnol 35:403419. doi:10.
1007/s10295-007-0300-y
3. Maru BT, Bielen AAM, Kengen SWM, Constant M, Medina F
(2012) Biohydrogen production from glycerol using Thermotoga
spp. Energy Procedia 29:300307. doi:10.1016/j.egypro.2012.09.
036
4. Maru BT, Bielen AAM, Constant M, Medina F, Kengen SWM
(2013) Glycerol fermentation to hydrogen by Thermotoga maritima: proposed pathway and bioenergetic considerations. Int J
Hydrogen Energy 38:55635572. doi:10.1016/j.ijhydene.2013.
02.130
5. Tan HW, Abdul Aziz AR, Aroua MK (2013) Glycerol production
and its applications as a raw material: a review. Renew Sustain
Energy Rev 27:118127. doi:10.1016/j.rser.2013.06.035
6. Wang C, Dou B, Chen H, Song Y, Xu Y, Du X, Zhang L, Luo T,
Tan C (2013) Renewable hydrogen production from steam
reforming of glycerol by NiCuAl, NiCuMg, NiMg catalysts. Int J Hydrogen Energy 38:35623571. doi:10.1016/j.ijhy
dene.2013.01.042
7. Lee PC, Lee WG, Lee SY, Chang HN (2001) Succinic acid
production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a
carbon source. Biotechnol Bioeng 72:4148. doi:10.1016/s01410229(98)00156-2
8. Papanikolaou S, Aggelis G (2002) Lipid production by Yarrowia
lipolytica growing on industrial glycerol in a single-stage continuous culture. Bioresour Technol 82:4349. doi:10.1016/
S0960-8524(01)00149-3
9. Papanikolaou S, Fick M, Aggelis G (2004) The effect of raw
glycerol concentration on the production of 1,3-propanediol by
Clostridium butyricum. J Chem Technol Biotechnol
79:11891196. doi:10.1002/jctb.1103
10. Wang K, Wang X, Xizhen G, Tian P (2012) Heterologous
expression of aldehyde dehydrogenase from Saccharomyces cerevisiae in Klebsiella pneumoniae for 3-hydroxypropionic acid
production from glycerol. Indian J Microbiol 52:478483. doi:10.
1007/s12088-012-0280-0
11. Li Y, Ge X, Tian P (2013) Gene arrangements in expression
vector affect 3-hydroxypropionic acid production in Klebsiella
pneumoniae. Indian J Microbiol 53:418424. doi:10.1007/
s12088-013-0390-3
12. Yang F, Hanna MA, Sun R (2012) Value-added uses for crude
glycerol-a byproduct of biodiesel production. Biotechnol Biofuels
5:13. doi:10.1186/1754-6834-5-13
13. Dharmadi Y, Murarka A, Gonzalez R (2006) Anaerobic fermentation of glycerol by Escherichia coli: a new platform for
metabolic engineering. Biotechnol Bioeng 94:821829. doi:10.
1002/bit.21025
14. Sharninghausen LS, Campos J, Manas MG, Crabtree RH (2014)
Efficient selective and atom economic catalytic conversion of

123

Indian J Microbiol

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

glycerol to lactic acid of glycerol to lactic acid. Nat Commun

5:5084. doi:10.1038/ncomms6084
Liu B, Christiansen K, Parnas R, Xu Z, Li B (2013) Optimizing
the production of hydrogen and 1,3-propanediol in anaerobic
fermentation of biodiesel glycerol. Int J Hydrogen Energy
38:31963205. doi:10.1016/j.ijhydene.2012.12.135
Patel SKS, Singh M, Kumar P, Purohit HJ, Kalia VC (2012)
Exploitation of defined bacterial cultures for production of
hydrogen and polyhydroxybutyrate from pea-shells. Biomass
Bioenergy 36:218225. doi:10.1016/j.biombioe.2011.10.027
Patel SKS, Kumar P, Kalia VC (2012) Enhancing biological
hydrogen production through complementary microbial metabolisms. Int J Hydrogen Energy 37:1059010603. doi:10.1016/j.
ijhydene.2012.04.045
Kumar P, Patel SKS, Lee JK, Kalia VC (2013) Extending the limits
of Bacillus for novel biotechnological applications. Biotechnol
Adv 31:15431561. doi:10.1016/j.biotechadv.2013.08.007
Patel SKS, Kalia VC (2013) Integrative biological hydrogen
production: an overview. Indian J Microbiol 53:310. doi:10.
1007/s12088-012-0287-6
Patel SKS, Kumar P, Singh M, Lee JK, Kalia VC (2015) Integrative approach to produce hydrogen and polyhydroxybutyrate
from biowaste using defined bacterial cultures. Bioresour Technol 176:136141. doi:10.1016/j.biortech.2014.11.029
Kivisto A, Santala V, Karp M (2010) Hydrogen production from
glycerol using halophilic fermentative bacteria. Bioresour Technol 101:86718677. doi:10.1016/j.biortech.2010.06.066
Lo YC, Chen XJ, Huang CY, Yuan YJ, Chang JS (2013) Dark
fermentative hydrogen production with crude glycerol from
biodiesel industry using indigenous hydrogen-producing bacteria.
Int J Hydrogen Energy 38:1581515822. doi:10.1016/j.ijhydene.
2013.05.083
Maru BT, Constanti M, Stchigel AM, Medina F, Sueiras JE
(2013) Biohydrogen production by dark fermentation of glycerol
using Enterobacter and Citrobacter sp. Biotechnol Prog
29:3138. doi:10.1002/btpr.1644
Sarma SJ, Brar SK, Le Bihan Y, Buelna G, Rabeb L, Soccol CR,
Naceur M, Rachid B (2013) Evaluation of different supplementary nutrients for enhanced biohydrogen production by Enterobacter aerogenes NRRL B 407 using waste derived crude
glycerol. Int J Hydrogen Energy 38:21912198. doi:10.1016/j.
ijhydene.2012.11.110
Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N (2005)
Hydrogen and ethanol production from glycerol-containing
wastes discharged after biodiesel manufacturing process. J Biosci
Bioeng 100:260265. doi:10.1263/jbb.100.260
Jitrwung R, Yargeau V (2011) Optimization of media composition for the production of biohydrogen from waste glycerol. Int J
Hydrogen Energy 36:96029611. doi:10.1016/j.ijhydene.2011.
05.092
Markov SA, Averitt J, Waldron B (2011) Bioreactor for glycerol
conversion into H2 by bacterium Enterobacter aerogenes. Int J
Hydrogen Energy 36:262266. doi:10.1016/j.ijhydene.2010.09.
090
Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R (2008) Fermentative utilization of glycerol by Escherichia coli and its
implications for the production of fuels and chemicals. Appl
Environ Microbiol 74:11241135. doi:10.1128/AEM.02192-07
Kivisto A, Santala V, Karp M (2011) Closing the 1,3-propanediol
route enhances hydrogen production from glycerol by Halanaerobium saccharolyticum subsp. saccharolyticum. Int J Hydrogen Energy 36:70747080. doi:10.1016/j.ijhydene.2011.03.012
Biebl H, Zeng AP, Menzel K, Deckwer WD (1998) Fermentation
of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella
pneumonia. Appl Microbiol Biotechnol 50:2429. doi:10.1007/
s002530051251

123

31. Ngo TA, Kim MS, Sim SJ (2011) High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana. Int J Hydrogen Energy 36:58365842. doi:10.1016/j.
ijhydene.2010.11.057
32. Reungsang A, Sittijunda S, Angelidaki I (2013) Simultaneous
production of hydrogen and ethanol from waste glycerol by
Enterobacter aerogenes KKU-S1. Int J Hydrogen Energy
38:18131825. doi:10.1016/j.ijhydene.2012.11.062
33. Liu F, Fang B (2007) Optimization of biohydrogen production
from biodiesel wastes by Klebsiella pneumoniae. Biotechnol J
2:374380. doi:10.1002/biot.200600102
34. Wu KJ, Lin YH, Lo YC, Chen CY, Chen WM, Chang JS (2011)
Converting glycerol into hydrogen, ethanol, and diols with a
Klebsiella sp. HE1 strain via anaerobic fermentation. J Taiwan
Inst Chem Eng 42:2025. doi:10.1016/j.jtice.2010.04.005
35. Selembo PA, Perez JM, Lloyd WA, Logan BE (2009) Enhanced
hydrogen and 1,3-propanediol production from glycerol by fermentation using mixed cultures. Biotechnol Bioeng
104:10981106. doi:10.1002/bit.22487
36. Sarma SJ, Brar SK, Le Bihan Y, Buelna G, Soccol CR (2014)
Mitigation of the inhibitory effect of soap by magnesium salt
treatment of crude glycerola novel approach for enhanced
biohydrogen production from the biodiesel industry waste. Bioresour Technol 151:4953. doi:10.1016/j.biortech.2013.10.042
37. Varrone C, Liberatore R, Crescenzi T, Izzo G, Wang A (2013)
The valorization of glycerol: economic assessment of an innovative process for the bioconversion of crude glycerol into ethanol and hydrogen. Appl Energy 105:349357. doi:10.1016/j.
apenergy.2013.01.015
38. Seifert K, Waligorska M, Wojtowski M, Laniecki M (2009)
Hydrogen generation from glycerol in batch fermentation process. Int J Hydrogen Energy 34:36713678. doi:10.1016/j.ijhy
dene.2009.02.045
39. Sarma SJ, Brar SK, Sydney EB, Le Bihan Y, Buelna G, Soccol
CR (2012) Microbial hydrogen production by bioconversion of
crude glycerol: a review. Int J Hydrogen Energy 37:64736490.
doi:10.1016/j.ijhydene.2012.01.050
40. Patel SKS, Purohit HJ, Kalia VC (2010) Dark fermentative
hydrogen production by defined mixed microbial cultures
immobilized on lignocellulosic waste materials. Int J Hydrogen
Energy 35:1067410681. doi:10.1016/j.ijhydene.2010.03.025
41. Patel SKS, Kumar P, Mehariya S, Purohit HJ, Lee JK, Kalia VC
(2014) Enhancement in hydrogen production by co-cultures of
Bacillus and Enterobacter. Int J Hydrogen Energy
39:1466314668. doi:10.1016/j.ijhydene.2014.07.084
42. Heyndrickx M, De Vos P, Vancanneyt M, De Ley J (1991) The
fermentation of glycerol by Clostridium butyricum LMG 1212t2
and 1213t1 and C. pasteurianum LMG 3285. Appl Microbiol
Biotechnol 34:637642. doi:10.1007/BF00167914
43. Fountoulakis MS, Manios T (2009) Enhanced methane and
hydrogen production from municipal solid waste and agroindustrial by-products co-digested with crude glycerol. Bioresour
Technol 100:30433047. doi:10.1016/j.biortech.2009.01.016
44. Kumar P, Singh M, Mehariya S, Patel SKS, Lee JK, Kalia VC
(2014) Ecobiotechnological approach for exploiting the abilities
of Bacillus to produce co-polymer of polyhydroxyalkanoate.
Indian J Microbiol 54:151157. doi:10.1007/s12088-014-0457-9
45. Kumar P, Pant DC, Mehariya S, Sharma R, Kansal A, Kalia VC
(2014) Ecobiotechnological strategy to enhance efficiency of
bioconversion of wastes into hydrogen and methane. Indian J
Microbiol 54:262267. doi:10.1007/s12088-014-0467-7
46. Porwal S, Kumar T, Lal S, Rani A, Kumar S, Cheema S, Purohit
HJ, Sharma R, Patel SKS, Kalia VC (2008) Hydrogen and
polyhydroxybutyrate producing abilities of microbes from
diverse habitats by dark fermentative process. Bioresour Technol
99:54445451. doi:10.1016/j.biortech.2007.11.011

Indian J Microbiol
47. Singh M, Kumar P, Patel SKS, Kalia VC (2013) Production of
polyhydroxyalkanoate co-polymer by Bacillus thuringiensis.
Indian J Microbiol 53:7783. doi:10.1007/s12088-012-0294-7
48. Kalia VC, Purohit HJ (2011) Quenching the quorum sensing
system: potential antibacterial drug targets. Crit Rev Microbiol
37:121140. doi:10.3109/1040841X.2010.532479
49. Naranjo JM, Posada JA, Higuita JC, Cardona CA (2013) Valorization of glycerol through the production of biopolymers: the
PHA case using Bacillus megaterium. Bioresour Technol
133:3844. doi:10.1016/j.biortech.2013.01.129
50. Singh M, Patel SKS, Kalia VC (2009) Bacillus subtilis as
potential producer for polyhydroxyalkanoates. Microb Cell Fact
8:38. doi:10.1186/1475-2859-8-38
51. Reddy SV, Thirumala M, Mahmood SK (2009) Production of PHA
and P (3HB-co-3HV) biopolymers by Bacillus megaterium strain
OU303A isolated from municipal sewage sludge. World J Microbiol Biotechnol 25:391397. doi:10.1007/s11274-008-9903-3
52. Reddy SV, Thirumala M, Mahmood SK (2009) A novel Bacillus
sp. accumulating poly (3-hydroxybutyrate-co-3-hydroxyvalerate)
from a single carbon substrate. J Ind Microbiol Biotechnol
36:837843. doi:10.1007/s10295-009-0561-8
53. Lopez JA, Naranjo JM, Higuita JC, Cubitto MA, Cardona CA,
Villar MA (2012) Biosynthesis of PHA from a new isolated
Bacillus megaterium strain: outlook on future developments with
endospore forming bacteria. Biotechnol Bioprocess Eng
17:250258. doi:10.1007/s12257-011-0448-1
54. Rohini D, Phadnis S, Rawal SK (2006) Synthesis and characterization of poly-b-hydroxybutyrate from Bacillus thuringiensis
R1. Indian J Biotechnol 5:276283
55. Cavalheiro J, De Almeida A, Grandfils C, Da Fonseca MMR
(2009) Poly(3 hydroxybutyrate) production by Cupriavidus necator using waste glycerol. Process Biochem 44:509515. doi:10.
1016/j.procbio.2009.01.008
56. Gozke G, Prechtl C, Kirschhofer F, Mothes G, Ondruschka J,
Brenner-Weiss G, Obst U, Posten C (2012) Electrofiltration as a
purification strategy for microbial poly-(3-hydroxybutyrate).

57.

58.

59.

60.

61.

62.

63.

64.

Bioresource Technol 123:272278. doi:10.1016/j.biortech.2012.

07.039
Reddy CSK, Ghai R, Kalia VC (2003) Polyhydroxyalkanoates:
an overview. Bioresour Technol 87:137146. doi:10.1016/S09608524(02)00212-2
de Almeida A, Giordano AM, Nikel PI, Pettinari MJ (2010)
Effects of aeration on the synthesis of poly (3-hydroxybutyrate)
from glycerol and glucose in recombinant Escherichia coli. Appl
Environ Microbiol 76:20362040. doi:10.1128/AEM.02706-09
Ashby RD, Solaiman DK, Foglia TA (2004) Bacterial poly (hydroxyalkanoate) polymer production from the biodiesel coproduct stream. J Polym Environ 12:105112. doi:10.1023/B:
JOOE.0000038541.54263.d9
Ashby RD, Solaiman DK, Strahan GD (2011) Efficient utilization
of crude glycerol as fermentation substrate in the synthesis of
poly (3-hydroxybutyrate) biopolymers. J Am Oil Chem Soc
88:949959. doi:10.1007/s11746-011-1755-6
Nikel PI, Pettinari MJ, Galvagno MA, Mendez BS (2008) Poly
(3-hydroxybutyrate) synthesis from glycerol by a recombinant
Escherichia coli arcA mutant in fed-batch microaerobic cultures.
Appl Microbiol Biotechnol 77:13371343. doi:10.1007/s00253007-1255-7
Full TD, Jung DO, Madigan MT (2006) Production of poly-bhydroxyalkanoates from soy molasses oligosaccharides by new,
rapidly growing Bacillus species. Lett Appl Microbiol
43:377384. doi:10.1111/j.1472-765X.2006.01981.x
Sangkharak K, Prasertsan P (2012) Screening and identification
of polyhydroxyalkanoates producing bacteria and biochemical
characterization of their possible application. J Gen Appl
Microbiol 58:173182. doi:10.2323/jgam.58.173
Sindhu R, Ammu B, Binod P, Deepthi SK, Ramachandran KB,
Soccol CR, Pandey A (2011) Production and characterization of
poly-3-hydroxybutyrate from crude glycerol by Bacillus sphaericus NII 0838 and improving its thermal properties by blending
with other polymers. Braz Arch Biol Technol 54:783794.
doi:10.1590/S1516-89132011000400019

123