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«Department of Chemical Engineering Università degli Studi di Napoli Federico II, Naples-Italy 2010 Department of Chemical Engineering Università ...»

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Conversion of Agro-industrial Wastes into Lipids

Suitable for Biodiesel Production

Thesis by

Abu Yousuf

In Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy in Chemical Engineering

XXIII Cycle

Tutor: Prof. Domenico Pirozzi

Department of Chemical Engineering

Università degli Studi di Napoli Federico II, Naples-Italy

2010

Department of Chemical Engineering

Università degli Studi di Napoli Federico II, Naples-Italy

Conversion of Agro-industrial Wastes into Lipids Suitable for Biodiesel Production by Abu Yousuf Tutor: Prof. Domenico Pirozzi Co-Tutor: Prof. Silvestro Crescitelli Prof. Colomba Di Blasi Course Coordinator: Prof. Pier Luca Maffettone Acknowledgements |i Acknowledgements I am extremely grateful to Almighty Allah for His blessing throughout my life.

I want to acknowledge my advisors, Prof. Domenico Pirozzi for his unconditional support and contribution to all my ideas. With his knowledge, guidance, kindness and patience I could develop the research assembled in this dissertation. I also would like to express my appreciation for their financial support through this research and my Ph.D. program.

I want to thank Prof. Silvestro Crescitelli and Prof. Colomba Di Blasi for their interest in serving the advisory committee and providing valuable suggestions and contributions. I want to recognize the scientific support from members of Prof. Greco‘s research group: Dr. Giuseppe Toscano and Dr. Maria Letizia Colarieti, their advice contributed to the experiments and instrumental support of this work.

I want to express gratitude to the PhD course coordinator Prof. Pier Luca Maffettone and Head of the Dept. of Chemical Engineering, Prof. Nino Grizzuti for their cooperation in all the administrative and academic activities related to this research.

Special thanks from the core of heart to Dr. Gaetano D Avino who introduced me the city, Naples and advised in every critical moment in Naples.

Thanks to the all current and past research students (Alessio, Francesco, Francesca, Amelia, Lisa, Pietro……..) of the laboratory of Biochemical Engineering for their friendship, I found a second family with them in foreign lands.

I want to thank my parents, specially my elder brother M A Hannan, and sister-in-law Rukshana Kader Popy for all their encouragement, affection and incredible support. Also thanks to my dear wife Sharmin Sultana for her wonderful love, inspiration, patience, and amazing complicity.

–  –  –

Microorganisms that can accumulate lipids at more than 20% of their dry mass are defined as oleaginous species. The majority of these lipids are triacylglycerol containing long-chain fatty acids, which are comparable to conventional vegetable oils. The recent, increasing interest towards the oleaginous microorganisms is due to the potential use of microbial triglycerides as feedstock for biodiesel production.

The oleaginous yeasts used in this thesis work appear to be very promising, due to their versatility, as they allow the use of different kinds of residues as nutrients. In particular, Lipomyces starkeyi is so far one of the best used, as it has been proved to store large amounts of lipids.

Lipomyces starkeyi were first grown in the presence of olive oil mill wastewaters (OMW), a medium difficult to process by biological treatments, due to the antimicrobial activities of their phenolic components. We demonstrated that Lipomyces can produce, without external organic supplements, a significant reduction of both the total organic carbon (TOC) and the total phenols content, leading to a significant increase of the germination index. The fatty acid distribution showed a prevalence of oleic acid, demonstrating the potential of L. starkeyi as a source of lipids to be used as a feedstock for the synthesis of II generation biodiesel. The performance of Lipomyces was improved by a preliminary dilution of OMW.

Lipomyces were able to grow also in the presence of wastewaters from cheese factory, leading to a satisfactory growth and to a significant reduction of the TOC levels.

Cellulosic agricultural residues were also evaluated as feedstock for oleaginous yeasts.

–  –  –

and seeds, at different nitrogen contents. The yeasts showed a favorable growth, with no need of addition of external nutrients.

Hydrolysates of Sorghum and Giant Reed were also studied as nutrients for the Lipomyces starkeyi. The conditions to maximize the lipid yield and the efficiency of the biomass conversion were found in terms of H2SO4 concentration (for the preliminary hydrolysis) and of medium composition (for the yeasts growth). Detoxification of hydrolysate with overlime and activated charcoal was carried out to reduce the concentration of microbial growth inhibitors, improving the growth of the yeasts in the undiluted hydrolysate.

In conclusion, the potential of oleaginous yeasts was demonstrated by the satisfactory microbial growth in the presence of different waste materials, and by the favorable composition of the triglycerides. Further studies are ongoing to optimize the preliminary hydrolysis of

–  –  –

ACKNOWLEDGEMENTS

Abstract

CONTENT





LIST OF FIGURES

LIST OF TABLES

LIST OF ABBREVIATIONS

CHAPTER-I: INTRODUCTION

1.1 Biorefineries

1.1.1 Biorefinery concept

1.1.2 Development of Biorefineries as an alternative to Petroleum refineries

1.1.3 Biorefineries versus alternative energies

1.1.4 Non-food agriculture

1.1.5 Biorefining as a new science

1.2 Biofuels

1.3 Methods for biodiesel production

1.3.1 First-generation biodiesel

1.3.2 Second-generation biodiesel

1.4 Oleaginous microorganisms

1.5 Objectives

CHAPTER-II: MATERIALS AND METHODS

2.1 Microorganisms and culture media

2.2 Olive Oil Mill Wastewater (OMW)

2.2.1 Fermentation with OMW

2.2.2 Phytotoxicity tests

Content |v 2.2.3 Statistical analysis

2.3 Lipid extraction and measurement

2.4 Fatty acids composition

2.5 Biomass analysis

2.6 Preparation of nutrient broth with cheesmaker wastewaters

2.7 Tomato waste hydrolysates (TWH)

2.7.1 Pre-adaptation of oleaginous yeasts.

2.7.2 Preparation of medium with hydrolysate

2.7.3 Fermentation

2.8 Pre-treatment (acid hydrolysis) of lignocellulosic biomass

2.9 Cellulose, hemicellulose and lignin measurement

2.10 Measurement of reducing sugar

2.11 Analysis of Microbial Biomass

2.12 Effect of Temperature

2.13 Detoxification of hydrolysate

2.14 Recycle of glycerin

CHAPTER-III: RESULTS AND DISCUSSION

3.1 Fermentation in synthetic medium

3.2 Olive-Mills Wastewater (OMW)

3.2.1 Economical impact of OMW

3.2.2 Fermentation in the presence of OMW

3.2.3 Logistic model for the biomass growth

3.2.4 Phytotoxicity

3.2.5 Lipid yield and composition

3.3 Cheesmaker wastewaters

3.3.1 Economical impact of CW

3.3.2 Sample specification

3.3.3 Cultures of Lipomyces in “acque di filature”

3.3.4 Culture of Lipomyces in diluted “acque di filature”

3.3.5 Cultures of Lipomyces in serum

C o n t e n t | vi

3.3.6 Cultures of Lipomyces in “scotta”

3.4 Lignocellulosic materials (LCM)

3.4.1 Economical impact of LCM

3.4.2 Pretreatment (Hydrolysis) of LCM

3.4.3 Detoxification

3.4.4 Liquid fermentation -Tomato waste hydrolysate (TWH)

3.4.4.1 Composition of Tomato waste

3.4.4.2 Effect of Nitrogen content

3.4.4.3 Effect of the dilution

3.4.4.4 Biomass and lipid yield

3.4.5 Sorghum (Sorghum bicolor)

3.4.5.1 Cultivation and Composition of Sorghum plant

3.4.5.2 Optimization of cultural medium

3.4.5.3 Growth kinetics

3.4.5.4 Nutrient consumption by L. starkeyi

3.4.5.5 Biomass and Lipid yields

3.4.6 Giant Reed Stem (Arundo donax)

3.4.6.1 Optimization of cultural medium

3.4.6.2 Effect of cultivation conditions

3.4.6.3 Effect of Temperature

3.4.6.4 Efficiency of detoxification methods

3.5 Recycle of glycerol as carbon source

3.5.1 Recover of the co-produced glycerol

3.5.2 Culture of Lipomyces starkey in the presence of glycerol

3.5.3 Culture of Yarrowia lipolytica in the presence of glycerol

3.5.4 Culture of Cryptococcus curvatus in the presence of glycerol

3.5.5 Lipid fraction obtained by the culture in the presence of glycerol

CONCLUSIONS

APPENDIX A

REFERENCES

–  –  –

Figure 1.1 Comparison of the basic-principles of the petroleum refinery and the biorefinery.

..... 1 Figure 1.2 Comparison of first, second generation biofuel and petroleum fuel (Naik et al.

, 2010)

Figure 1.3 Short flow chart of experimental activity

Figure 2.1 Olive mill waste water

Figure 2.2 Dry biomass cultured in OMW, the source of lipid

Figure 2.3 Tomato Squeezers, and tomato waste (seed and peel), after extraction of pulp.

........ 34 Figure 3.1 Growth kinetics of four oleaginous yeasts using an N-limiting synthetic medium in batch reactors. Operating conditions: T = 30°C, 160 rpm, medium composition as in the Method paragraph.

Figure 3.2 Growth of Lipomyces starkeyi using an N-limiting synthetic medium in batch reactors, under multiple additions of the nitrogen source.

Operating conditions: T = 30°C, 160 rpm, medium composition as in the Method paragraph.

Figure 3.3 Olive oil production in EU

Figure 3.4 Growth of Lipomyces starkeyi in the presence of olive mill wastewaters (OMW) in batch reactors.

Operating conditions: T = 30°C, 160 rpm. OMW composition as in the Method paragraph

–  –  –

Figure 3.6 Comparison of experimental measurements of biomass concentration (X, g/L) and TOC (g/L) with the theoretical data obtained with the logistic model, with reference to the culture of L.

starkeyi in batch cultures, in the presence of raw OMW.Op....... 52 Figure 3.7 Diagram of mozzarella, cheese and dairy production with sampling points............... 58 Figure 3.8 Evolution of TOC of the ‗acque di filatura‘ during the culture of L. starkeyi............ 60 Figure 3.9 Cell growth in the medium of ‗acque di filatura‘ during the culture of L. starkeyi.... 60 Figure 3.10 Evolution of TOC of the medium (diluted and undiluted) of ‗acque di filatura‘ during the culture of L. starkeyi

Figure 3.11 Cell growth in the diluted medium of ‗acque di filatura‘ during the culture of L.

starkeyi

Figure 3.12 Evolution of TOC of the medium (diluted and undiluted) of ‗siero‘ during the culture of L.

starkeyi

Figure 3.13 Evolution of TOC (a) and cell growth (b) of the medium of ‗scotta‘

Figure 3.14 Effect of concentration of H2SO4 on hydrolysis process at condition-121C, 20 min

Figure 3.15 Optimization of acid hydrolysis.

Figure 3.16 Formation of inhibitors during hydrolysis of lignocellulosic materials (adapted from Palmqvist and Hahn-Hagerdal, 2000).

Figure 3.17 Composition of tomato waste (% dry weight basis)

Figure 3.18 Effect of N content on the culture of Lipomysis sterkeyi in the broth of TWH.

....... 79 Figure 3.19 Effect of dilution on the culture of Lipomysis sterkeyi in the broth of TWH............ 80

–  –  –

Figure 3.21 Composition of stem of Sorghum and their distribution to hydrolysate and residual fraction (values on basis 10)

Figure 3.22 Optimization of cultural medium (Sorghum) in terms of dilution

Figure 3.23 Change of reducing suagr during the growth of L.

starkeyi in the medium of Sorghum hydrolysate

Figure 3.24 Growth kinetics of microorganism in cellulosic hydrolysate

Figure 3.25 Change of C sources with microbial growth of L.

starkeyi in SGM

Figure 3.26 Microbial biomass, cultured on hydrolysate of lignocellulosic materials.

................ 89 Figure 3.27 Optimization of cultural medium of hydrolysate of Giant Reed Stem in terms of dilution

Figure 3.28 Effect of cultivation condition on cell growth of L.

starkeyi

Figure 3.29 Change of C sources with microbial growth of L.

starkeyi in GRS

Figure 3.30 Growth profile of L.

starkeyi at temp. 15˚C, 20˚C, 30˚C and fluctuated (15-30˚C). 94 Figure 3.31 Growth of microorganisms on different Detoxified medium (WT-, OL, AC, OLAC)

Figure 3.32 Growth of L.

starkeyi in the detoxified mediums

Figure 3.33 Growth profile of Lipomyces starkeyi using glycerol as the sole carbon source.

... 100 Figure 3.34 Growth profile of Yarrowia lipolytica using glycerol as the sole carbon source.... 101

–  –  –

List of Tables Table 1.1 The agro-industrial residues and their fermentation process to accumulate lipid by oleaginous microorganism

Table 1.2 Oleaginous microorganisms with their lipid accumulation efficiency from residual materials

Table 2.1 Composition of the olive mill wastewater.

Table 3.1 Sugars, proteins and phenols removal obtained during the Lipomyces starkeyi culture in the presence of olive mill wastewaters.

Table 3.2 Comparison of Experimental Values of the Growth Parameters with the Theoretical Data Obtained with the Logistic Model, with Reference to the Culture of L.

starkeyi in Batch Reactors, under Different Experimental Conditions

Table 3.3 Germination index of Latuca sativa seeds on untreated and treated OMW samples.



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