Bioconversion of Crude Glycerol into Microbial Lipid and its Subsequent Conversion to Polyol

Bijaya Kumar Uprety* & Sudip K. Rakshit

Biorefining Research Institute (BRI)Lakehead University

International Forest Biorefining Conference (IFBC); May 9-11, 2017

Contents

• Research Background

• Research Problems & Objectives

• Method Outline

• Results

• Conclusion

1

Research Background

2

Introduction to Biodiesel

• Cleaner burning substitute

• Common feedstocks

Plants oil

Animal fats

• Blended with diesel – 2 to 20%(v/v)

Fig 1. Biodiesel cycle

3

Biodiesel production process

• Transesterification of triglycerides

• Common alcohol - methanol

• Commonly catalyst - sodium

hydroxide

• Product- biodiesel

• By product- crude glycerol

Fig 2. Transesterification of triglyceride into biodiesel

Fig 3. Overall biodiesel production from triglycerides

4

Crude glycerol as a byproduct from biodiesel industries

• 10 wt.% of glycerol produced

• Expected production by 2020;

Biodiesel- 37.9 billion liters

Crude glycerol- 3.7 billion liters

• Contains impurities and requires further purification for commercial application

Glut of glycerol due to rise in biodiesel production

Fig. 4 Biodiesel and crude glycerol production across the world (OECD-2016)

5

Need of crude glycerol valorization

• Fate of crude glycerol

High grade glycerol

Animal feedstock

Low energy fuel

• Bioconversion of crude glycerol to various products e.g. Hydrogen, propanoic acid, succinic acid, citric acid, etc.

• For sustainable biodiesel production

• To manage crude glycerol in environmental friendly manner

1. Ciriminna, R., Pina, C. Della, Rossi, M., & Pagliaro, M. (2014). Understanding the glycerol market. European Journal of Lipid Science and Technology, 1432–1439

Uneconomical

6

Bioconversion of crude glycerol to microbial lipid

• One possible route- microbial lipids production

• Lipids chemically similar to plant based oils

• Potential feedstock for biodieseland polyol (precursor for PU foams)

• Advantages of such conversion:

Easy integration

Add annual revenues

Manage crude glycerol

7

Fig 5. Bioconversion of crude glycerol to microbial lipid and subsequent conversion of the latter to biodiesel

Oleaginous yeast

Biodiesel Crude glycerol

Microbial lipids

Polyol

Crude glycerol to microbial lipid: literature survey

• Low yields due to impurities

• Microbial lipid for biodieselproduction recommended

• Microbial lipid to polyol not studied yet

Yeast StrainsBiomass

Conc.(g/L)

Lipid Content (wt.%)

Lipid Conc.(g/L)

References

Yarrowia lipolytica QU 21 3.85 22.1 0.85Kiran et al.

2013

Trichosporondoidesspathulata JU4-57

13.8 56.4 7.75Kitcha & Cheirship

2013

Candida sp. LEB-M3 19.7 50.2 9.88Duarte et al. 2013

Trichosporon fermentansCICC 1368

16.0 32.4 5.18Liu et al.

2016

Trichosporon cutaneumAS 2.0571

17.4 32.2 5.60Liu et al.

2016

Kodamaea ohmeri BY4-523

10.3 53.3 5.49Kitcha & Cheirship

2013

Table 1. Biomass and lipid production by previously reported yeasts strains growing oncrude glycerol as carbon source

Conversion of microbial lipids to polyol will (1) Expand its application (2) Make more sustainable

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Research Rationale&

Objectives

9

Research problems

• Robust strain to produce large amount of microbial lipid from crude glycerol is required

• Potential use of microbial lipids for polyol production yet to be explored

10

Research objectives

• Production of microbial lipid from crude glycerol using robust oleaginous yeast

• Conversion of microbial lipid to polyol and its comparison with polyols from other vegetable oils

• Demonstrate the use of microbial oil based polyol for the production of polyurethane foams

11

Method Outline

12

Overall method

1. Saifuddin, N., Chun Wen, O., Wei Zhan, L., Xin Ning, K., 2010. Palm oil based polyols for polyurethane foams application2. Comparative study on total lipid determination using Soxhlet, Roese-Gottlieb, Bligh & Dyer, and modified Bligh & Dyer extraction methods3. Guo, A., & Ivan, J.&, Petrovic, Z., 2000. Rigid polyurethane foams based on soybean oil. J. Appl. Polym. Sci. 77, 467–473

30 °C, 300 rpm, 1.2 vvm, pH 6, 7 days

Crude glycerol

Batch fermentation (1L) Microbial oil

Epoxidation and ring opening

Polyols

RBD palm oil Canola oil

Biomass dried & extracted using modified Bligh-Dyer method

As per method reported by Saifuddin et al (2010)• Epoxidation- peroxy acid (H2O2 + formic acid)

• Ring opening- phosphoric acid

As per Guo et al (2000)Reacted with Toluene Di-

isocyanate (TDI), –CNO/–OH molar ratio of 1.1

30 𝟎C, 200 rpm, pH 6, 7 days

R. toruloides ATCC 10788

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Results

14

Characterization of crude glycerol

Components Wt.%

Glycerol 44.56

Methanol 13.86

Water 10.74

Soap 32.97

FAMEs 4.38

Free Fatty Acids

0.48

Ash 10.74

Fig 7. Composition of crude glycerol used

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Fig 6. Crude glycerol from biodiesel industry

Fermentation conditions

Fermentation type

Biomass Conc. (g/L)

Lipid Conc. (g/L)

Lipid content (%

wt.)

In pure glycerol(PG media)

Flask 11.86±1.85 4.62±0.69 39.01±5.86

In pure glycerol with 15 g/L of

methanol(GM media)

Flask 9.78±1.76 3.36±0.38 34.38±3.93

In crude glycerol(CG media)

Flask 23.63±2.92 13.86±1.48 58.66±6.26

In crude glycerol(CG media) Batch 27.48±0.93 18.69±1.46 68.03±5.30

Table 2. Biomass concentration, lipid concentration and lipid content obtained by growing R.toruloides ATCC 10788 for 7 days in different media containing 48.2 g/L of glycerol

Growth of R. toruloides in pure and crude glycerol

• Methanol- Growth and lipid inhibited

• Three times more lipidproduced using crude glycerol

• Biodiesel traces positively influenced lipid production

• Soap was being consumed

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Fatty acid profile of obtained microbial lipid

Oil sourceC16:0

(%)

C18:0

(%)

C18:1

(%)

C18:2

(%)

C18:3

(%)

Others

(%)

SFA

(%)

UFA

(%)References

Palm 42.70 2.13 39.37 10.62 0.21 4.97 ~49 ~51(Zambiazi et

al., 2007)

Canola 3.75 1.87 62.41 20.12 8.37 3.48 ~7 ~93(Zambiazi et

al., 2007)

Corn 10.34 2.04 25.54 59.27 1.07 1.74 ~13 ~87(Zambiazi et

al., 2007)

Sunflower 6.20 2.80 28.0 62.2 0.16 0.64 ~9 ~91(Orsavova et

al., 2015)

Rapeseed 4.60 1.70 63.3 19.6 1.20 9.60 ~6 ~94(Orsavova et

al., 2015)

R. toruloides

ATCC 1078824.39 16.38 47.16 12.05 - - ~ 40 ~ 60 This study

Table 3. Fatty acid profile of varieties of oils used in this study. SFA: Saturated fatty acid; UFA: Unsaturated fatty acid

The unsaturation level of the obtained oil was similar to palm oil

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FT-IR and NMR analysis results

Fig 8. Comparison of FT-IR analysis of microbial oil (MO),epoxidized (EMO) and its polyol (MOP)

FT-IR and NMR result showed complete conversion of oil to polyol

Fig 9. 1H proton NMR of microbial oil (MO), epoxidizedmicrobial oil (EMO) and microbial oil polyol (MOP)

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Hydroxyl value of microbial oil based polyol

Table 4. Comparison of hydroxyl value of microbial oil based polyol with other vegetable oils based polyol obtained via

epoxidation and oxirane ring opening

Oil type Ring opening Agent usedHydroxyl value(mg KOH/g of

sample)

References

Canola Methanol 173.6 (Zlatanic et al., 2004)

Palm oil Hexamethylene glycol 110 (Pawlik and Prociak, 2012)

Palm oil Phthalic acid 70-80 (Ang et al., 2014)

Soybean oil HCl 197 (Guo et al., 2000b)

Soybean oil 1,2-propanediol 289.31 (Dai et al., 2009)

Soybean oil HBr 182 (Guo et al., 2000b)

Linseed oil Methanol 247.8 (Zlatanic et al., 2004)

Sunflower oil Methanol 177.8 (Zlatanic et al., 2004)

Rapeseed oil Diethylene glycol 114-196 (Rojek and Prociak, 2012)

Canola oil Phosphoric acid 266.86 This study

Palm oil Phosphoric acid 222.32 This study

Microbial oil Phosphoric acid 230.30 This study

OH value of obtained microbial lipid was similar to polyol obtained using vegetable oils

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Conversion of polyols to polyurethane foams

20

Polyols

Toluene Di-isocyanate (TDI)

Sl. No. Chemical Role of chemical Mass of chemicals (g)

1 Polyol Monomer 100

2 DBTDL Main catalyst 1

3 Tegostab B-8404 Surfactant 2

4 DMEA Co-catalyst 1

5 Water* Blowing agent 2

Fig 10. Conversion of polyols to polyurethane foams

Table 5. The polyol formulation used to mix with toluene di-isocyanate (TDI) to

maintain an isocyanate index of 1.1 to produce polyurethanes ( Guo et al. 2000)

Conclusions

• R. toruloides ATCC 10788 can withstand high amount of impurities in crude glycerol

• Lipid concentration improved when crude glycerol was used

• Production of polyol from microbial lipid demonstrated

• Suitability of microbial based polyol for polyurethane production studied

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Future work

• Hemicellulose to polyurethane foams can be produced using this strain

• Characterization of produced polyurethane foams

• Production of special types of polyurethanes

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Uprety, B.K., Reddy, J.V., Dalli, S.S., Rakshit, S.K., 2017. Utilization of microbial oil obtained from crude glycerol for the production of polyol and its subsequent conversion to polyurethane foams. Bioresour. Technol. 235, 309–315. doi:10.1016/j.biortech.2017.03.126

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Acknowledgments

All BRI MEMBERS&

DR. SUDIP RAKSHIT’S TEAM

Thank you for listening !

Any Questions?