Bálint Balázs

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Utilization of protein-rich animal waste materials to produce biohydrogen   Ph.D. Thesis    Written by:  Balázs Bálint    Supervisors:  Prof. Kornél L. Kovács  Dr. Gábor Rákhely    Ph.D. School in Biology  Institute of Biophysics, Biological Research Center,  Hungarian Academy of Sciences, and Department of Biotechnology, Faculty of Science and Informatics, University of Szeged   Szeged, Hungary 2008  2 Introduction   Today, the generation of electricity, heating and transportation highly depend on fossil fuels.  Combustion of natural gas, oil and coal releases enormous quantities of carbon dioxide, which is considered as the major cause of global warming and climate change.  In addition, the formation of fossil fuels is much slower than their consumption rate and their depletion is expected within a couple of decades.  Concerns about the limited availability of fossil fuels and their negative effect on the environment urge the scientific community to seek for clean energy carriers that can be produced from renewable sources.  Hydrogen is among the best candidates since it burns to clean water vapor with zero carbon dioxide emission.  It can be generated from many renewable energy sources including solar energy, hydropower, wind power and biomass.    A great number of microorganisms are known to produce hydrogen and the possible use of microbes for hydrogen production is extensively studied.  Photosynthetic cyanobacteria, green algae and purple bacteria are potential candidates for solar energy driven biohydrogen production while fermentative bacteria and archaea are well suited for fermentative hydrogen evolution using cheap organic substrates.   Agriculture and related industries produce large quantities of by-products, which are rich in carbohydrates or peptides.  Currently, these wastes are mainly disposed or decomposed through expensive procedures.  In principle, these organic waste materials could be used to cover the biomass requirement of fermentative hydrogen-producing facilities.  The combination of biological waste decomposition and fermentative biohydrogen production is economically very promising strategy as it can solve two problems at the same time.  It offers an environmentally sound, cheap alternative for the treatment of agricultural wastes and additionally it produces environmentally energy carriers replacing fossil fuels.  3 Aims of the study  The aim of the present study was the development of a novel biological waste utilization system that combines microbial degradation of protein-rich animal waste materials (feathers, pig hair, meat meal) with the production of a useful product, biohydrogen.   1. Construction of a two-stage fermentation system to utilize  keratinaceous wastes for biohydrogen production. • Evaluation of chicken feathers, digested by  B. licheniformis KK1, as a carbon and energy source for dark hydrogen fermentation.  Testing the ability of potential known hydrogen producer strains to utilize keratin hydrolysate for hydrogen evolution. • Optimization of the keratin degradation step to produce a fermentation broth that is ideal for the next hydrogen production step. • Optimization and scaling-up of the hydrogen-producing fermentation step. • Testing the two-stage system with additional keratin wastes  (i.e. goose feathers, pig hair). • Determination of overall conversion yields for different keratinaceous wastes. 2. Adaptation of the established two-stage system for meat meal. • Evaluation of meat meal as a nutrient for dark hydrogen  fermentation. • Scaling-up of the hydrogen production step on meat meal  hydrolysate. • Determination of overall conversion yield for meat meal.  4 3. Enhancing the waste degradation step using molecular biology  techniques. • Cloning and sequencing the keratinase gene from  B. licheniformis KK1. • Heterologous expression of the keratinase in E. coli.  Methods   Hydrolysis of chicken feathers was carried out with B. licheniformis KK1 both in Erlenmeyer flasks and in 1 L glass vessels of a Braun Biostat DCU 3 fermenter.  Feather degradation was followed by monitoring the changes in protein concentration and protein pattern of the fermentation broth over time.  Hydrolysates obtained from the bacterial decomposition of feather waste were added to nutrient-stripped minimal media and tested in hydrogen producing fermentations with potential hydrogen evolving microorganisms (Caldicellulosiruptor saccharolyticus, Escherichia coli K12, Thermococcus litoralis and Pyrococcus furiosus).  Microbial hydrogen production was followed by gas chromatography.  In addition to chicken feather hydrolysate, fermentation broths prepared from goose feathers, pig hair and meat meal were also evaluated as possible nutrient sources for microbial hydrogen production.  Scale-up studies on the hydrogen-producing fermentations with T. litoralis were performed in a 6.9 L batch fermenter.  The keratinase gene of B. licheniformis KK1 was amplified via high fidelity PCR and its nucleotide sequence was determined.  DNA manipulations and analyses were performed according to standard techniques and the manufacturer's recommendations.  For heterologous expression of the kerA in  E. coli a gene expression cassette was constructed based on the pBAD/gIII system.  Keratinase activity was assayed spectrofotometrically using the chromogenic peptide substrate N-succinyl-Ala-Ala-Pro-Phe-pNA.  5 Results  My results are summarized in the following points:  1. I have developed a minimal medium (CMSY) and methodology for the evaluation of numerous organic materials as nutrient sources for hyperthermophilic hydrogen-producing microorganisms.  2. Decomposition of chicken feather was performed in shaken Erlenmeyer-flasks using the Bacillus licheniformis KK1 strain, and I have proven that near complete degradation of feather occurs within 84 hours of incubation, which was accompanied by accumulation of small-sized peptides in the fermentation broth.  I have determined the degradation time optimal for the concomitant hydrogen-producing fermentation.  3. I have evaluated several potential hydrogen-producing microorganisms (Escherichia coli, Caldicellulosiruptor saccharolyticus T. litoralis and Pyrococcus furiosus) and demonstrated Thermococcus litoralis to be the best candidate for the hydrogen-producing fermentation on keratin hydrolysate.  4. I have disclosed that milling is required for chicken feather fermentation carried out with stirring. I have achieved a 3.5-times scale-up of the keratin fermentation under well-controlled conditions. Monitoring the keratin degradation process in fermenter I have shown that the feather meal disappeared within 138 hours of incubation and, in paralel, peptides were accumulated in the fermentation broth.    6 5. I have proven that feather hydrolysate is a well-suited nutrient for  T. litoralis comparable to the expensive commercial peptidic substrate, Bacto Peptone.  6. I have carried out a 125 times scale up of the hydrogen production step on feather hydrolysate using a high temperature fermenter. I have shown that the highest hydrogen concentration and the best overall conversion yield on feather hydrolysate can be achieved when the hydrogen fermentation step is carried out in fermenter.  7. I have adapted the two-stage fermentation system for the utilization of additional substrates including goose feather, pig hair and meat meal. I have thus proven that it is possible to combine the decomposition of numerous animal waste materials with the production of biohydrogen.  8. I have isolated the kerA gene coding for the keratinase in B. licheniformis KK1 and determined its nucleotide sequence.  9. I have created a protein overexpression construct for the production of the keratinase in E. coli. I have detected the presence of active keratinase in the induced periplasmic fraction of E. coli cells transformed with pBLK-BAD.  7 Publications related to the thesis  Balázs Bálint, Zoltán Bagi, András Tóth, Gábor Rákhely, Katalin Perei, and Kornél L. Kovács Utilization of keratin-containing biowaste to produce biohydrogen. Appl Microbiol Biotechnol, 2005, 69:404-410  Balázs Bálint: Biohydrogen production from keratin-containing animal wastes, Acta Biol Szeged 2006, 50(3-4):137  K. Bélafi-Bakó D. Búcsú. Z. Pientka,  B. Bálint, Zs. Herbel, K.L. Kovács, M. Wessling Integration of biohydrognen fermentation and gas separation processes to recover and enrich hydrogen.  Int J Hydrogen Energ, 2006, 31:1490-1495  B. Bálint, Z. Bagi, A. Tóth, K. Perei, K. L. Kovács and G. Rákhely Biohydrogen production from keratin-containing wastes Proceedings of HYPOTHESIS VI (Hydrogen Power Theoretical and Engineering Solutions International Symposium p. 411-422 (2005).  B. Bálint, A. Tóth, G. Rákhely, K. Perei, Z. Bagi, B. Pónya, K. L. Kovács: Production of pure hydrogen from feather waste, Days of Chemical Engineering, Veszprém, 2004   Patents registered in Hungary related to the thesis  B. K. Bélafi, L. Gubicza, D. Búcsú, F. Molnár, L. Kiss, Z. Pientka, K. L. Kovács, G. Rákhely, B. Bálint, Zs. Herbel: Membrane process for recovery and concentration of biohydrogen, P0500581, 2005  8 B. Bálint, A. Tóth, G. Rákhely, K. Perei, Z. Bagi, B. Pónya, K. L. Kovács:  Microbiological process for decomposing keratinaceous wastes, biomass obtained thereof and use of biomass as medium for microorganisms, P0203998, 2002  Other publications  Zoltán Bagi, Norbert Ács, Balázs Bálint, Lenke Horváth, Krisztina Dobó, Katalin R. Perei, Gábor Rákhely and Kornél L. Kovács: Biotechnological intensification of biogas production. Appl Microbiol Biotechnol, 2007, 76:473-482  R. Horváth, T. Orosz B. Bálint, M. Wessling, G. H. Koops, G. C. Kapantaidakis, K. Bélafi-Bakó: Application of gas separation to recover biohydrogen produced by Thiocapsa roseopersicina, Desalination, 2004, 163:261-265  K. L. Kovács, Á. T. Kovács, G. Maróti, Z. Bagi, G. Csanádi, K. Perei, B. Bálint, J. Balogh, A. Fülöp, L. S. Mészáros, A. Tóth, R. Dávid, D. Latinovics, A. Varga and G. Rákhely: Improvement of biohydrogen production and intensification of biogas formation. Rev. Environ. Sci. Biotechnol, 2004, 3:321-330  K. Perei, Z. Bagi, B. Bálint, Gy. Csanádi, P. Hofner, L. Horváth, Gy. Kardos, M. Magony, G. Rákhely, Gy. Román, A. Tóth, Sz. Zsíros and K. L. Kovács: Environment biotechnological utilization of microbes, Biokémia, 2004, 28:54-58. (article in Hungarian)      9 Acknowledgements   I wish to express my sincere thanks to:   My supervisors  Professor Kornél L. Kovács and Dr. Gábor Rákhely   All the Ph.D. students, diploma workers and colleagues especially   Dr. András Tóth, Dr. Katalin Perei, Zoltán Bagi, Zsófia Herbel Dr. Mária Takács, Dr. László Galgóczi and  Dr. Rui Miguel Mamede Branca  I am grateful to my parents and my brother for their support and encouragement.  I wish to say thank you for my wife Boglárka for her understanding that writing the Ph.D thesis was a tense period of life. 

Utilization of protein-rich animal waste materials to produce biohydrogen

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Utilization of protein-rich animal waste materials to produce biohydrogen

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