a report of the proposed student project program (biofuels ... · biodiesel derived crude glycerol...

GULBARGA UNIVERSITY

Department Of Post Graduate Studies and Research in Biotechnology

Jnana Ganga, Kalaburagi-585106.

A Report of the proposed Student Project Program (Biofuels)

“Bioconversion of Biodiesel derived Crude Glycerol

to Polyhydroxyalkanoates (PHA/PHB)”

Submitted by

KOMAL TIMANE

SHOBHA GUDUR

VISHNU JADHAV

Research Guide (s)

PROF. G. R. NAIK Department of Biotechnology

Gulbarga University

Kalaburagi

PRAMOD BHIMRAO KULKARNI Department of Biotechnology

Gulbarga University

Kalaburagi

Submitted to

KARNATAKA STATE COUNCIL FOR SCIENCE AND TECHNOLOGY

Bengaluru

2016

Supported by

KARNATAKA STATE BIOENERGY DEVELOPMENT BOARD

Bengaluru

Project Reference No: 39S_B_MSC_008

Email: [email protected] Phone: (O) +91 – 08472 263290 (R) +91 – 08472 245337

GULBARGA UNIVERSITY, KALABURAGI POST GRADUATE DEPARTMENT OF STUDIES AND RESEARCH IN BIOTECHNOLOGY

JNANA GANGA – KALABURAGI – 585 106 – KARNATAKA – INDIA

Certificate This is to certify that, the project work entitled “Bioconversion of

Biodiesel derived Crude Glycerol to Polyhydroxyalkanoates (PHA/PHB)”

submitted by Ms. TIMANE KOMAL SANJAY, Ms. GUDUR SHOBHA

DATTATRAY & Mr. VISHNU JADHAV, Submitted to Karnataka State

Council for Science and Technology, Bengaluru, under the STUDENT

PROJECT PROGRAM (BIOFUELS), Supported by Karnataka State

Bioenergy Development Board, Bengaluru, for the completion of the

project. The project work is carried out by them in the Department of

Biotechnology, Gulbarga University, Kalaburagi.

Place: Kalaburagi Research Supervisor

Date: Prof. G. R. Naik

Prof. G. R. NAIK Department of Biotechnology

Gulbarga University,

Kalaburagi – 585 106

mailto:[email protected]

Declaration

We hereby declare that the present report entitled “Bioconversion of

Biodiesel derived Crude Glycerol to Polyhydroxyalkanoates (PHA/PHB)”

Submitted to Karnataka State Council for Science and Technology,

Bengaluru, under the Student Project Program (Biofuels), Supported by

Karnataka State Bioenergy Development Board, Bengaluru, is the result of

the project work carried out under the guidance of Prof. G. R. Naik & Mr.

Pramod Bhimrao Kulkarni, Department of Biotechnology, Gulbarga

University, Kalaburagi.

We further declare that results of this work have not been previously

submitted for any other degree or discipline.

Place: Kalaburagi

Date:

Ms. TIMANE KOMAL SANJAY

Ms. GUDUR SHOBHA DATTATRAY

Mr. VISHNU JADHAV

Acknowledgement

We would like to express our hearty and sincere gratefulness to The Almighty for having

blessed us to undertake the project work in Biofuels and to commence and successfully complete

the same by His grace.

We immensely express our special gratitude and deep sense of esteem to our project

supervisor’s Dr. G. R. Naik, Professor Department of Biotechnology, Gulbarga University,

Kalaburagi & Mr. Pramod Bhimrao Kulkarni, Department of Biotechnology, Gulbarga

University, Kalaburagi for their constant encouragement throughout the project whose timely

and valuable guidance without that the undertaken task would not have been successfully

accomplished.

We extend our gratitude to Dr. Ramesh Londonkar, Professor and Chairman, Dr.

Kelmani Chandrakant R., Professor and Dr. M. B. Sulochana, Associate Professor, Department

of Biotechnology, Gulbarga University, Kalaburagi who have helped us throughout the project

work for their valuable suggestions and generous help during the work.

We express our gratitude to Karnataka State Council for Science and Technology,

Bengaluru for providing an opportunity to work on the task by accepting our proposal under the

STUDENT PROJECT PROGRAM (BIOFUELS).

We are thankful to Karnataka State Bioenergy Development Board, Bengaluru, for

providing financial assistance required to carry out the project.

We shall be failing in our duty if we do not place on record our hearty and loving

gratefulness to our loving parents, family and friends for their constant inspiration without which

we could not have completed our task to the subjective satisfaction.

Ms. TIMANE KOMAL SANJAY

Ms. GUDUR SHOBHA DATTATRAY

Mr. VISHNU JADHAV

Contents

SI No. Title Page No.

1 Introduction 1-3

2 Review of literature 4-17

3 Materials and Methods 18-20

4 Results 21-23

5 Summary and Conclusion 24

References 25-39

Bioconversion of Biodiesel derived Crude Glycerol to Polyhydroxyalkanoates (PHA/PHB)

Department of Biotechnology, Gulbarga University, Kalaburagi. SPP Ref No: 39S_B_MSC_008

Introduction



1. Introduction

Biodiesel production has increased exponentially over the years; leading to the large crude

glycerol generation obtained by the transesterification of vegetable oils. There are wide range of

applications of pure glycerol in food, pharmaceuticals, cosmetics and many other industries. It is

very cost effective to refine crude glycerol to a high purity especially for the small and medium

biodiesel producers. Also the increasing amount of crude glycerol is causing storage problem and

environmental hazard. Many research studies have been taken up with innovative ideas finding

alternative utilization of crude glycerol. One such alternative is to use it as carbon source for

PHA/PHB/PHB production.

Accumulation of nondegradable plastic in the environment is one of the major causes of

pollution nowadays. Indian Supreme court made a statement stating “Plastic bags threat is more

serious than the atom bomb. Plastic bags photo-degrade; over time they breakdown into smaller,

more toxic petropolymere which eventually contaminate soils and waterways. As a consequence

microscopic particles can enter the food chain (National Geographic news Sept 2, 2003). The

effect on wildlife can be catastrophic, birds become terminally entangled, and nearly 200 different

species of sea life including whales, dolphins, seals and turtles die due to feeding on plastic

accumulated in the aquatic habitat which is mistaken for food (World life fund report 2005).

Considering the tremendous threats caused by the plastic, there is a need to search for the

alternative which can replace this plastic. One such alternative is to use the Eco friendly bioplastic

(Ployhydroxyalkanoates/Polyhydroxybutyrates) produced using renewable substrates and which

are ecofriendly. Bioplastics are biobased biodegradable plastics with almost similar properties to

synthetic plastics. Biodegradation can be explained as a chemical process during microorganisms

that present in the environment convert materials into natural substances such as water, carbon



dioxide and compost. The term biobased means the material is partly derived from biomass.

Synthetic plastics remain in the environment for long time as they are resistant to degradation

(Aminabhavi et al, 1990). Bioplastics are made from variety of sources like polysaccharides,

lipids and also proteins (Avrous, 2004; Hernandez- and Krochta, 2008; Siracusa et al, 2008;

Gonzalez et al, 2009). So there are number of substrates which can be used for the

Polyhydroxyalkanoate/Polyhydroxybuterate (PHA/PHB) production out of which the crude

glycerol will be particularly focused carbon source in the present work

The PHA bioaccumulation trait is widespread among the bacteria & archaeal domains

with PHA producing microbes occurring in more than 70 bacterial & archael genera (Tappel,

Nomura, 2009; Poli et al, 2011). Bioaccumulated PHA is stored in the form of intracellular lipid

granules in these microbes (Galia, 2010). Acting as biocatalysts these PHA producing

microorganisms enable the coupling of myriad of carbon catabolic pathways together with PHA

anabolic pathways, there by playing a key role the diversification of PHA production from

various carbon sources. these carbon sources include saccharides (e.g. Fructose, Maltose, Lactose,

Xylose, Arabinose etc), n-alkanes (e.g. hexane, octane, dodecane etc), n-alkanoic acids (e.g.

acetic acid, propionic acid, butyric acid, valeric acid, lauic acid, oleic acid etc), n-alcohols (e.g.

methanol, ethanol, octanol, glycerol etc), & gases, such as methane & carbon dioxide (Anderson,

and Dawes, 1990; Verlinden et al, 2007). Waste streams which provide a free source of carbons

have also been identified for PHA production (Koller et al, 2010). These include waste frying oil,

vinegar waste, waste fats, food waste, agricultural waste, domestic waste water, plant oil mill

effluent, crude glycerol from biodiesel production, plastic waste, land fill gas etc.

Recently, much work has been done using biodiesel- derived waste glycerol for PHA

production by Cupriavidus neactor JMP 134, Paracoccus denitrificans (Mothes et al., 2007),



Cuprividus neactor DSM.545 (Cavalheiro et al., 2009), Bacillus sonorensis, Halomonas

hydroyhermalis (Shrivastav et al., 2010), Halomonas sp. KM-1 (Kawata and Aiba., 2010),

Osmophilic organisms (Koller et al., 2005), Pseudomonas oleovorans NRRL B-14682 and

Pseudomonas corrugate 388 (Ashby et al., 2004) from different sources of biodiesel feedstock

(Jantima et al, 2010).

Looking into the problems associated with synthetic plastics and biodiesel derived crude

glycerol, advantages of biodegradable plastics/ bioplastics over synthetic plastics and the

potentiality of microorganisms in utilizing biodiesel derived crude glycerol and producing the

bioplastic, the present work was undertaken with following objectives.

1. Collection of marine samples.

2. Isolation of glycerol utilizing organisms.

3. Screening for PHA/PHB producers using biodiesel derived crude glycerol as carbon

source.

4. Quantification of PHA/PHB from screened isolates.



Review of Literature



2. Review of Literature

Crude Glycerol:

The principle byproduct of biodiesel production is the crude glycerol, which is 10% wt of

vegetable oil (Dasari et al, 2005). Such crude glycerol possesses very low value because of the

impurities. As the demand and production of biodiesel grows, the quantity of crude glycerol

generated will be considerable high. Further refining of the crude glycerol will depend on the

economy of production scale and/or the availability of glycerol purification facility. Larger scale

biodiesel producers refine their crude glycerol and move it to markets in other industries. It is

generally treated and refined through filtration, chemical additions, and fractional vacuum

distillation to yield various commercial grades. If it is used in food, cosmetics and drugs, further

purification is needed such as bleeching, deodoring, and ion exchange to remove trace properties.

Purifying it to that stage however is costly and generally out of the range of economic feasibility

for the small to medium sized plants.

Chemical compositions of crude glycerol

The chemical composition of crude glycerol mainly varies with the type of catalyst used to

produce biodiesel, the transesterification (Fig no 1) efficiency and recovery efficiency of the

biodiesel, other impurities in the feedstock, and whether the methanol and catalysts were

recovered. All of these considerations contribute to the composition of the crude glycerol fraction.

For instance, Hansen et al (2009) studied the chemical compositions of 11 crude glycerol

collected from 7 Australian biodiesel producers and indicated that the glycerol content ranged

between 38% and 96%, with some samples including more than 14% methanol and 29% ash.

Such variations would be expected with small conversion facilities. In most cases, biodiesel



production involves the use of methanol and a homogeneous alkaline catalyst, such as sodium

methoxide and potassium hydroxide. Accordingly, methanol, soap, Catalysts, salts, non-glycerol

organic matter, and water impurities usually are contained in the crude glycerol. Crude glycerol

obtained from sunflower oil biodiesel production process had the following composition (w/w):

30% glycerol, 50% methanol, 13% soap, 2% moisture, approximately 2-3% salts (primarily

sodium and potassium), and 2-3% other impurities (Asad et al, 2008). Moreover, while the same

feedstocks were employed, the crude glycerol from alkali- and lipase-catalyzed transesterification

contained different purities of glycerol (Mu et al, 2006). The salt content in crude glycerol, from

biodiesel production via homogeneous alkaline catalysts, ranged from 5% to 7% which makes the

conventional purification techniques more costly. Heterogeneous processes using enzymes and

solid metal-oxide catalysts have been promoted as good alternatives to homogeneous alkaline

catalysts in terms of improving the quality of crude glycerol. However, even in heterogeneous

transesterification processes, impurities existing in the natural raw feedstocks tend to accumulate

in the glycerol phase. Therefore, purification of crude glycerol is required, in most cases, to

remove impurities in order to meet the requirements of existing and emerging uses.

Fig no1: Transesterification reaction.



Value-added opportunities for crude glycerol

Worldwide, crude glycerol derived from biodiesel conversion has increased from 200, 000

tons in 2004 (Pagliaro and Rossi, 2008) to 1.224 million tonnes in 2008 (Biodiesel 2020, Market

Survey). Meanwhile, the global market for refined glycerol was estimated to be roughly 900, 000

tons in 2005 (Nilles, 2006). Therefore, it is of great importance for scientists to find new

applications for refined and crude glycerol. Recently, numerous papers have been published on

direct utilization of crude glycerol from biodiesel production. In the following sections, detailed

discussions on utilization of crude glycerol are presented.

Animal feedstuff

Using glycerol as a feed ingredient for animals dates back to the 1970’s (Fisher et al,

1973). However, glycerol's utilization in feeds has been limited by the availability of glycerol

(Kerr et al, 2007). Recently, the possibilities of using crude glycerol from biodiesel in feeds have

been investigated because of the increase in the price of corn and the surplus of crude glycerol.

Crude glycerol in non-ruminant diets

Glycerol has high absorption rates and is good energy source. Once absorbed, it can be

converted to glucose for energy production in the liver of animals by the enzyme glycerol kinase

(Kerr et al, 2007). Crude glycerol samples, from different biodiesel producers, were analyzed as

energy sources. The digestible energy (DE) values for 85% of the crude glycerol samples were in

the range of 14.9-15.3 MJ/kg with metabolizable energy (ME) values in the range of 13.9-14.7

MJ/kg (Dasari, 2007). Crude glycerol was an excellent source of calories for non-ruminants, for

example, the ME determined in broilers, laying hens and swine were 15.2, 15.9 and 13.4 MJ

ME/kg, respectively (Kerr et al, 2007). In growing pigs and laying hens, 14.0 MJ/kg apparent DE



(Lammers et al, 2008) and 15.9 MJ/kg nitrogen-corrected apparent ME (AMEn) (Lammers et al,

2007) were reported, respectively, which implied that crude glycerol was used efficiently. The

AMEn of crude glycerol was metabolized efficiently by broiler chickens with an AMEn of 14.4

MJ/kg. That was very similar to the general energy (GE) of 15.2 MJ/kg (Dozier et al, 2008). In

nursery pigs, the GE concentration of crude glycerol depended on the concentration of glycerol,

methanol, and fatty acids, with ME as a percent of GE averaging 85.4% (Kerr et al, 2009).

Although crude glycerol can be added to animal feed, excess glycerol in the animal diet

may affect normal physiological metabolism. A few manuscripts have been published that

focused on the levels of crude glycerol fed and the performance of crude glycerol in animal feeds.

The improvement of daily gains by pigs depended on the actual intake of glycerol during the

growing period but not on the finishing period. The dietary treatments had no significant effects

on meat quality (Kijora et al, 1996; Schieck et al, 2010) . When crude glycerol was added to the

diets of weaned pigs, at levels up to 10%, the feed performance was enhanced (Shields et al,

2011). Up to 9% crude glycerol could be added to the diets of lactating sows with performances

similar to sows fed standard corn-soybean meal control diets (Schieck et al, 2010). No detrimental

effects, with respect to egg performance, egg quality, nutrient retention, and metabolizable

energy, were found when crude glycerol was incorporated at a level of 6% in the diet of laying

hens (Swiatkiewicz and Koreleski, 2009). In broiler diets, increasing the intake level of crude

glycerol increased feed conversion ratio but did not affect growth performance and nutrient

digestibility (McLea et al, 2011). Crude glycerol was used effectively at levels of 2.5 or 5%. But

the use of 10% crude glycerol resulted in poor feed flow. The influences of levels and quality of

crude glycerol on pellet quality needs further study (Cerrate et al, 2006). Crude glycerol, added at

up to 15% dry matter in the diets of finishing lambs, could improve feedlot performance,



especially during the first 14 d, and had no associated effect on carcass characteristics (Gunn et al,

2010). Compared to medium-quality hay, diets for meat goats with up to 5% crude glycerol

proved to be beneficial (Hampy et al, 2008). In addition, the inclusion of purified glycerol at up to

15% of the dry matter ration of lactating dairy cows was possible, without deleterious effects on

feed intake, milk production, and yield (Donkin, 2008, Donkin et al, 2009). When crude glycerol

was added at levels of 8% or less, based on dry matter in cattle finishing diets, it improved weight

gain and feed efficiency (Parsons et al, 2009). Apart from the above mentioned investigations, a

patent described approaches for using or incorporating crude glycerol into animal feeds as well as

feeding recommendations (Cecava et al, 2008). In all, the use of crude glycerol as an animal feed

component has great potential for replacing corn in diets, and is gaining increasing attention.

However, one must be aware of the presence of potential hazardous impurities in crude glycerol

from biodiesel. For example, residual levels of potassium may result in wet litter or imbalances in

dietary electrolyte balance in broilers (Cerrat et al, 2006). The levels of methanol must be

minimized because of its toxicity (Kerr et al, 2007, Lammers et al, 2008, Cerrate et al, 2006,

Donkin et al, 2009). More attention should be paid to the crude glycerol from small scale

biodiesel facilities that use simple batch distillation or evaporation techniques.

Feedstock’s for chemicals produced via biological conversions

1, 3-propanediol:

The anaerobic fermentative production 1, 3-propanediol is the most promising option for

biological conversion of glycerol. Mu et al. (2006) demonstrated that crude glycerol could be

used directly for the production of 1, 3-propanediol in fed-batch cultures of Klebsiella

pneumoniae. The differences between the final 1, 3-propanediol concentrations were small for

crude glycerol from the methanolysis of soybean oil by alkali- (51.3 g/L) and lipase-catalysis (53



g/L). This implied that the composition of crude glycerol had little effect on the biological

conversion and a low fermentation cost could be expected. Further, the production of 1,3-

propanediol by K. pneumoniae was optimized using response surface methodology. The

maximum yield of 1, 3-propanediol was 13.8 g/L (Oh et al, 2008). More recently, the production

of 1, 3-propanediol, from crude glycerol from Jatropha biodiesel by K. pneumoniae ATCC 15380,

was optimized. The obtained 1, 3-propanediol yield, purity and recovery were 56 g/L, 99.7% and

34%, respectively (Hiremath et al, 2011). Additionally, an incorporated bioprocess that combined

biodiesel production by lipase with microbial production of 1, 3-propanediol by K.

pneumoniaewas developed in a hollow fiber membrane. The bioprocess avoided glycerol

inhibition on lipase and reduced the production cost (Mu et al, 2008). Clostridium butyricum also

could be used to produce 1, 3-propanediol from crude glycerol. For example, C. butyricum VPI

3266 was able to produce 1, 3-propanediol from crude glycerol on a synthetic medium. Trivial

differences were found between commercial glycerol and crude glycerol (González et al,

2004). C. butyricum strain F2b and C. butyricum VPI 1718 potentially could convert crude

glycerol to 1, 3-propanediol (Papanikolaou and Aggeli, 2003, Papanikolaou, 2008; Chatzifragkou

2011). In order to avoid the isolation of 1,3-propanediol from crude glycerol fermentation media,

a one vessel bio- and chemo-catalytic process was developed to convert crude glycerol to

secondary amines directly in a biphasic system without intermediate separation of 1, 3-

propanediol (Liu et al, 2009). Additionally, Chatzifragkou et al. (2010) studied the effects of

different impurities in crude glycerol on 1, 3-propanediol production by C. butyricum. The double

bond from long-chain fatty acids or methyl esters might influence the growth performance of the

microorganism, methanol did not affect the microbial conversion and the presence of NaCl had

certain effect during a continuous process but not a batch process.



Citric acid:

A few reports are available on the use of crude glycerol for citric acid biosynthesis.

The production of citric acid from crude glycerol by Yarrowia lipolytica ACA-DC 50109 was not

only similar to that obtained from sugar-based conventional media (Papanikolaou and Aggelis,

2003) but also single-cell oil and citric acid were produced simultaneously (Papanikolaou et al

2008, Papanikolaou and Aggelis, 2009). When a fed-batch fermentation by acetate-negative

mutants of Y. lipolytica Wratislavia AWG7 strain was used to ferment crude glycerol, the final

concentration of citric acid was 131.5 g/L, similar to that obtained from pure glycerol (139 g/L).

On the other hand, when Y. lipolytica Wratislavia K1 was used, a lower concentration of citric

acid (about 87-89 g/L) and a high concentration of erythritol (up to 47 g/L) were obtained

(Rywińska et al, 2009). It was in line with the results shown by Rymowicz W et al. (2008).

Further, Y. lipolytica Wratislavia K1 proved to be superior to other strains by producing erythritol

and not citric acid from crude glycerol under optimal conversion conditions, which may be a

valuable development (Rymowicz et al, 2009). Y.lipolytica LGAM S (7) 1 also showed potential

for converting crude glycerol to citric acid (Papanikolaou et al, 2002). More recently, it was

reported that Y. lipolytica N15 could produce citric acid in high amounts, specifically, up to 98

g/L of citric acid and 71 g/L of citric acid were produced from pure glycerol medium and crude

glycerol medium, respectively (Kamzolova et al, 2011).

Hydrogen and other lower molecule fuels

The bacterium Rhodopseudomonas palustris was capable of photofermentative conversion

of crude glycerol to hydrogen. Nearly equal productions were obtained from crude glycerol and

pure glycerol. Up to 6 moles H2 per mole glycerol were obtained, which was 75% of theoretical.

Both rates and yields of hydrogen production could be modified by changing the concentration of



added nitrogen. However, some technical obstacles, such as enhancing the efficiency of light

utilization by the organisms and developing effective photobioreactors, still need to be solved

during development of a practical process (Sabourin and Hallenbeck, 2009). When Enterobacter

aerogenes HU-101 was employed, hydrogen and ethanol were produced at high yields and with

high production rates. But the crude glycerol should be diluted with a synthetic medium in order

to increase the rate of glycerol utilization (Ito et al, 2005). For maximizing hydrogen production,

Jitrwung and Yargeau (2011), optimized some media compositions of E. aerogenes ATCC 35029

fermented crude glycerol process. More recently, it was reported that K.pneumoniae mutant strain

and nonpathogenic Kluyvera cryocrescens S26 were promising for producing ethanol from crude

glycerol (Choi et al, 2011; Oh et al, 2011). In addition, crude glycerol, as a co-substrate, could be

used to enhance hydrogen and especially methane production during the anaerobic treatment of

different feedstocks including the organic fraction of municipal solid wastes, sewage sludge and

slaughterhouse wastes (Fountoulakis and Manios, 2009, Fountoulakis et al, 2010; López et al,

2009 ).

Polyunsaturated fatty acids:

Docosahexaenoic acid:

A series of papers on the production of docosahexaenoic acid (DHA)-rich algae

were published, using crude glycerol, by fermentation of the alga Schizochytrium limacinum. For

supporting alga growth and DHA production, 75-100 g/L concentration of crude glycerol was

recommended as the optimal range. The algal DHA yield was influenced significantly by

temperature and ammonium acetate concentration. The optimal amounts for temperature and

ammonium acetate were 19.2°C and 1.0 g/L, respectively. The highest DHA yield obtained was

4.91 g/L under the optimized culture conditions (Chi et al, 2007). Different sources of crude



glycerol did not result in significant variations in algal biomass compositions. The resulting algae

had a similar content of DHA and a comparable nutritional profile to commercial algal biomass.

That proposed good potential for using crude glycerol-derived algae in ɷ-3-fortified foods or

feeds (Pyle et al, 2008). Further, DHA-containing algae have been developed as replacements for

fish oil for ɷ-3 fatty acids. Crude glycerol was used to produce fungal biomass that served as

eicosapentaenoic acid (EPA)-fortified foods or feeds through fungal fermentation with

fungus Pythium irregulare. Growing in medium containing 30 g/L crude glycerol and 1.0 g/L

yeast extract, the EPA yield and productivity could reach 90 mg/L and 14.9 mg/L per day,

respectively. The resulting EPA content was low compared to microalgae for EPA. Optimizing

culture conditions and developing high cell density culture techniques are imperative in future

work (Athalye et al, 2009). Recently, it was reported that continuous culture was an effective

approach for studying the growth kinetics and behaviors of the algae on crude glycerol (Ethier et

al, 2011).

Lipids

As the sole carbon source, crude glycerol could be used to produce lipids which might be a

sustainable biodiesel feedstock. For example, crude glycerol could be used for

culturing Schizochytrium limacinum SR21 and Cryptococcus curvatus. S. limacinum algal growth

and lipid production were affected by the concentrations of glycerol. Higher concentrations of

glycerol had negative effects on cell growth. For batch culturing of crude glycerol derived from

yellow grease, the optimal glycerol concentrations for untreated and treated crude glycerol were

25 and 35 g/L, respectively. With 35 g/L, the obtained highest cellular lipid content was 73.3%.

Methanol remaining in crude glycerol could harm S. limacinum SR21 growth (Liang et al, 2010).

For C. curvatusyeast, fed-batch was a better process than batch for lipid production. Culturing for



12 days, the lipid content from one-stage fed-batch operation and two-stage fed-batch process

were 44.2% and 52%, respectively. Methanol did not have significant inhibitory effect on cell

growth. The produced lipid had high concentration of monounsaturated fatty acid and was good

biodiesel feedstock (Liang et al, 2009).

Further, Saenge et al, (2011) presented that oleaginous red yeast Rhodotorula

glutinis TISTR 5159, cultured on crude glycerol, produced lipids and carotenoids. The addition of

ammonium sulfate and Tween 20 increased the accumulation of lipids and carotenoids. When

fed-batch fermentation was employed, the highest lipid content, lipid yield and carotenoids

production were 10.05 g/L, 60.7% and 6.10 g/L, respectively. Chlorella protothecoides also

converted crude glycerol to lipids. The lipids yield was 0.31 g lipids/g substrate (O'Grady and

Morgan, 2011). Similarly, with C. protothecoides and crude glycerol (62% purity), Chen and

Walker (2011) demonstrated that the maximum lipid productivity of 3 g/L per day was obtained

in a fed-batch operation, which was higher than that produced by batch process. Additionally,

Chatzifragkou et al. (2011) studied the potential of fifteen eukaryotic microorganisms to convert

crude glycerol to metabolic products. The results showed that yeast accumulated limited lipids

(up to 22 wt %, wt/wt, in the case of Rhodotorula sp.), while fungi accumulated higher amounts

of lipids in their mycelia (ranging between 18.1 and 42.6%, wt/wt, of dry biomass).

Poly hydroxyalkanoates:

Poly hydroxyalkanoates (PHA) represent a complex class of naturally occurring bacterial

polyesters and have been recognized as good substitutes for non-biodegradable petrochemically

produced polymers. Ashby et al. (2004) reported that crude glycerol could be used to produce

PHA polymer. PHB is the most-studied example of biodegradable polyesters belonging to the

group of PHA. The study of the feasibility of using crude glycerol for PHB production,



with Paracoccus denitrificans and Cupriavidus necator JMP 134, showed that the resulting

polymers were very similar to those obtained from glucose. But the PHB production decreased

significantly when NaCl-contaminated crude glycerol was used. The authors suggested that the

harmful effect of the NaCl-contaminant could be reduced by mixing crude glycerol from different

manufacturers (Mothes et al, 2007). Further, a process based on the Cupriavidus necator DSM

545 fermentation of crude glycerol was designed for the large-scale production of PHB. However,

sodium still hindered the cell growth (Cavalheiro et al, 2009). Zobellella denitrificans MW1 could

utilize crude glycerol for growth and PHB production to high concentration, especially in the

presence of NaCl. Therefore, it was recommended as an attractive option for large-scale

production of PHB with crude glycerol (Ibrahim and Steinbüchel, 2009). Additionally, when

mixed microbial consortia (MMC) was used for PHA production from crude glycerol, it was

found that methanol in the crude glycerol was transformed to PHB by MMC. Further, it was

estimated that a 10 million gallon per year biodiesel plant would have the potential of producing

20.9 ton PHB (Dobroth, 2011). More recent report showed at Pseudomonas oleovorans NRRL B-

14682 could also be used for PHB production from crude glycerol (Ashby et al, 2004).

PHAs are the most versatile fully biodegradable polymers with properties similar to

conventional plastics (Steinbu & Fuchtenbusch, 1998). Other biodegradable polymers such as

chemically synthesized plastics (e.g. polyglycollic acid and polylactic acid) and starch-based

plastics (e.g. starch-polyethylene) have also appeared on the scene but they lack variability in

structure and extensive material properties. Appreciable number of PHAs with more than 150

monomers (Steinbuchel & Lutke, 2003) has been identified with molecular masses ranging from

50,000 to 1,000,000 Da. As PHAs are biodegradable and immunologically inert, they have



promising future applications, particularly in medical related fields, despite their expensive

production.

Structure and properties of PHAs

PHAs are thermoplastic or elastomeric polyesters (polyoxoesters) of R-hydroxyalkanoic

acid (HA) monomers (Fig no 2) that are biosynthesized by a wide range of Gram-positive and

Gram-negative bacteria as intracellular carbon and energy storage compounds (Anderson &

Dawes, 1990; Lee, 1996). In most cases, they are produced and accumulated under stressed

conditions such as nitrogen, phosphorous or oxygen limitation (Dawes, 1990; Lefebvre et al,

1997; Ryu et al, 1997; Shang et al, 2003) with excess carbon sources. Structurally, these polymers

are classified on the basis of the number of carbon atoms that range from 4 to 14 (Madison &

Huisman, 1999; Taguchi & Doi, 2004) and the type of monomeric units, producing

homopolymers or heteropolymers. PHAs with 3–5 carbon atoms are considered as short chain

length PHAs (scl-PHAs). Examples of this class include poly (3-hydroxybutyrate), P (3HB) and

poly (4-hydroxybutyrate), P(4HB)). Medium chain length PHAs (mcl-PHAs) contains 6-14

carbon atoms. Examples include homopolymers poly (3-hydroxyhexanoate), P(3HHx), poly(3-

hydroxyoctanoate), P(3HO) and heteropolymers such as P(3HHx-co-3HO) (Anderson & Dawes

1990). While PHAs with C4 homopolymers were first to be appreciated industrially and are

relatively better characterized compared to other PHAs, more recently reported PHAs are superior

in their versatility. P (3HB) has high molecular weight and crystallinity with melting point of

180oC and an elongation to break of 5% (Holmes, 1988; Hahn, 1994; Lee et al, 2000).

Specifically designed copolymers have been used to eliminate or reduce the brittleness and

thermal instability of polyhydroxybutyrate However, medium chain length PHAs and their

copolymers have low crystallinity (20–40%) and do not break easily (extension to break of 300–



450%). They behave as elastomers and their composition can be manipulated for a range of

applications (Anderson & Dawes, 1990).

Fig no 2: General structure of polyhydroxyalkanoates. R1 and R2 are alkyl groups

(C1–C13)

Applications

Application of PHAs, both in quantity and type has expanded particularly over the past 2–3

decades. Earlier applications were mainly in the areas of packaging e.g. cosmetic containers,

shampoo bottles (Hocking & Marchessault, 1994; Weiner, 1997), cover for cardboards and papers,

milk cartons and films, moisture barriers in nappies and sanitary towels (Hocking & Marchessault,

1994; Lauzier et al, 1993), pens, combs, bullets (Chen, 2005; Chen, & Qiong, 2005) and bulk

chemical production using depolymerised PHA (Brandl et al, 1988; Lee et al, 1999; Su et al, 2000;

Lee et al, 2000). Patents related to a range of application of PHAs in molding, containers, pens,

golf tees, diapers, personal hygiene materials, hot-melt and pressure-sensitive adhesives, films,

flavour delivery agents in foods, dairy cream substitutes, fabrics and materials for manufacturing

compostable articles and solvents have been reviewed by (Madison & Huisman, 1999). More

recently attention has focused on the medical applications of PHAs. This includes their usage as

cardiovascular products (pericardial and atrial septal repair patches, scaffolds for regeneration of

arterial tissues, vascular grafts, cardiovascular stents and heart valves), prodrugs; and their efficacy

in nerve and soft tissue repair, dental and maxillofacial treatment (guiding tissue and bone



regeneration), drug delivery (tablets, implants, micro-carriers), nutrition (both for man and

animal), orthopaedic and urology procedures and wound management (sutures, dusting powders,

dressings).

Other chemicals

Beyond the chemicals mentioned above, several other processes for producing useful

chemicals from crude glycerol via biotransformations have been developed. A continuous

cultivation process and a recently isolated bacterium Basfia succiniciproducens DD1 were

identified for succinic acid production. The process was characterized as having great process

stability, attractive production cost, and impossible pathogenicity of the production strain, but the

final production strain needs to be examined further for commercial succinic acid production

(Scholten et al, 2009). Via simulation method, Vlysidis et al. (2011) showed that the succinic acid

co-production from crude glycerol, for a 20 years biodiesel plant, would improve the profit of the

overall biorefinery by 60%. Further, crude glycerol, as the sole carbon source, had the potential of

producing phytase in industrial scale in high cell density fermentations with recombinant Pichia

pastoris possessing a pGAP-based constitutive expression vector (Tang et al, 2009) and

producing butanol with Clostridium pasteurianum. The highest yield of butanol was 0.30 g/g,

which was significantly higher than the 0.15-0.20 g/g butanol yield typically obtained during the

fermentation of glucose using Clostridium acetobutylicum. However, further understanding and

optimizing of the process are still needed. It remained unclear what impact the impurities in crude

glycerol would have on the solvent formation (Taconi et al, 2009). Similarly, crude glycerol could

be used in a bioprocess with P. pastoris without any purification. Canola oil-derived crude

glycerol was the most favorable carbon source and showed great potential for the production of

additional value-added products such as the recombinant human erythropoietin and cell growth



(Çelik et al, 2008). Crude glycerol also could be economic carbon and nutrient sources for

bacterial cellulose (BC) production. The BC amount obtained was about 0.1 g/L after 96 h

incubation. The addition of other nutrient sources (yeast extract, nitrogen and phosphate) to crude

glycerol culture media increased the BC production by ~200% (Carreira et al, 2011).

Additionally, Gluconobacter sp. NBRC3259 could be used to produce glyceric acid from

crude glycerol with an activated charcoal pretreatment. 49.5 g/L of glyceric acid and 28.2 g/L

dihydroxyacetone were produced from 174 g/L of glycerol (Habe et al, 2009).

When Staphylococcus caseolyticus EX17 was employed, crude glycerol could be used for solvent

tolerant lipase production (Volpato et al, 2008). More recently, it was reported that Ustilago

maydis was a good biocatalyst for converting crude glycerol to glycolipid-type biosurfactants and

other useful products (Liu et al, 2011). Fungal protein, Rhizopus microsporus var. oligosporus,

production on crude glycerol was another potential use of crude glycerol. The obtained fungal

biomass contained high amounts of threonine and could be co-fed with commercial sources. But

feeding formulation need be further studied (Nitayavardhana and Khanal, 2011).



Materials and Methods



3. Materials and Methodology

Materials:

All the chemicals were purchased from Himedia India Ltd & SD Fine Chemicals, India

and glassware’s from Borosil India Ltd.

Methodology:

1. Collection of marine soil samples and Biodiesel derived Crude Glycerol:

The Biodiesel Derived Crude Glycerol was obtained from Biofuel Information and

Demonstration Centre, Gulbarga University, Kalaburagi, the marine soil samples were collected

from coastal areas of Mumbai (Aksa beach, Juhu beach and Chaupati beach), Goa (Condolium

beach, Colangutta beach, Baga beach, Anjuna beach and Vaga toura beach) & Karwar, Karnataka

(Tagore beach, Dev bhag and beach, Karwar port beach) Gokaran, Karnataka (Gokarna beach

and Om beach), Murudeshwar, Karnataka(Murudeshwar beach), Udupi, Karnataka (Malpe

beach).

Pretreatment of Crude Glycerol:

Crude glycerol from Biofuel Information and Demonstration Centre, Gulbarga University,

was derived from the alkali catalysed transesterification process. The obtained glycerol hence

contained major amount of sodium hydroxide & methanol, which are detrimental to the microbial

growth. Hence, the pretreatment was be done by 1) the excess methanol content was removed by

distillation process. 2) The initial pH of crude glycerol was found to be 12, which was then

brought down to neutral pH using 0.1N HCL; this process converts the unreacted free fatty acids

into insoluble fatty acids, forming clumps & was removed by filtrating. 70% Glycerol was



obtained by this pretreatment, which was further used as supplement carbon source for isolation

of marine organisms.

2. Isolation of Crude Glycerol Utilizing Marine bacteria:

The marine bacterium utilizing glycerol were isolated using the serial dilution technique in

aseptic conditions, further through sub-culturing axenic cultures were obtained. Sea water agar

medium was used with supplement of 1% (W/V) pure glycerol and crude glycerol respectively,

Sea water agar medium constituted of Peptone- 5 g/l, yeast extract- 5 g/l, Beaf extract- 3 g/l,

Agar- 15 g/l & Synthetic Sea water- 1000ml. The constituents of Synthetic sea water are NaCl- 24

g/l, MgSo4.7H2O- 7 g/l, MgCl2.6H2O- 5.3 g/l, KCl- 0.7 g/l, CaCl2- 0.1 g/l.

3. Screening for PHA/PHB producing isolates:

Primary screening:

The obtained isolates were further screened for their potentiality to produce PHA/PHB

using staining techniques like Sudan Black B and Nile Blue-A staining (Ostle and Holt, 1982).

The Sudan Black B stain, a lipophilic stain used to stain the lipid granules, helping in

differentiating the PHA/PHB producers and non-producers. The Nile Blue, which particularly

stains the PHA/PHB granules within the cell illuminating bright orange florescence under UV

light of 460nm wavelength

Sudan Black B staining:

Sudan Black B stain was prepared by dissolving 0.3gm of Sudan Black B dissolved in 75

ml of 95% ethanol and making volume to 100ml by distilled water. Heat fixed samples were

stained with Sudan Black Solution for 10 minutes; excess stain was clarified using Xylene and

blot dried. Further counter stained with 0.5% aqueous Safranine.



Nile Blue A staining:

1% aqueous solution of Nile Blue A was prepared and filtered before use. Isolates were

cultured on sea water agar supplemented with 1% (v/v) crude glycerol and stained with Nile blue

solution for 10 minutes. Excess stain was drained off; plates were irradiated with UV light to

observe the orange fluorescence.



Results



4. Results

2. Isolation of Marine organisms utilizing biodiesel derived crude glycerol

Based on colony morphology 35 different isolates were obtained on Sea Water Agar

medium supplemented with 1% (V/V) crude glycerol. These were sub cultured and maintained as

axenic cultures. The obtained isolates were coded as SKV 1 to SKV 35. (Fig no 3)

Fig no. 3: Marine isolates grown on sea water medium supplemented with 1% (v/v) crude

glycerol

3. Screening of PHA/PHB producing isolates:

3.1. Primary screening:

Sudan Black B staining revealed the presence of lipid granules (PHA/PHB) within the

obtained 35 isolates. (Fig no. 4) When further screened with Nile Blue A stain. 10 isolates

exhibited the Fluorescence under UV light confirming the presence of PHA/PHB within these 10

isolates. (Table No. 1, Fig no 5)



Table No. 1: Results of Screening using Nile Blue A.

Sl. No Isolates Result1 SKV1 -2 SKV2 -3 SKV3 -4 SKV4 +5 SKV5 +6 SKV6 +7 SKV7 _8 SKV8 _9 SKV9 +10 SKV10 _11 SKV11 -12 SKV12 +13 SKV13 -14 SKV14 -15 SKV15 -16 SKV16 +17 SKV17 -18 SKV18 -19 SKV19 -20 SKV20 +21 SKV21 -22 SKV22 -23 SKV23 -24 SKV24 +25 SKV25 -26 SKV26 -27 SKV27 -28 SKV28 +29 SKV29 -30 SKV30 -31 SKV31 -32 SKV32 -33 SKV33 +34 SKV34 -35 SKV35 -



Fig no 4: Micrograph showing the dark stained cell with Sudan Black B

Fig no 5: A: Negative result for PHA/PHB production by isolates. B. Positive result for

PHA/PHB production by isolates.

A B



Summary and Conclusion



5. Summary and Conclusion

Global interest in biodiesel as an alternative have increased during past years, which is

usually produced by transesterification of tree borne oil in presence of an alcohol and a strong

base, generating crude glycerol as a primary by product. As the biodiesel production has

increased over the years has led to increased production of crude glycerol, which is in impure

form containing large amount of methanol and sodium hydroxide which causes environmental

hazard. Hence, narrowing its commercial applications.

The present work was taken up with an objective to find out an alternative usage of crude

glycerol by using it as a carbon source for isolating the marine organisms and further converting it

to the PHA/PHB. Thus providing a value added market to the crude glycerol. Successfully

isolated 35 different isolates which could utilize the crude glycerol as carbon source, further

screening with sudan black and nile blue staining revealed 10 isolates to be potent in converting

the crude glycerol to the PHA/PHB.



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a report of the proposed student project program (biofuels ... · biodiesel derived crude glycerol...

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