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5Статья поступила в редакцию 07.08.12. Ред. рег. № 1393
The article has entered in publishing office 07.08.12. Ed. reg. No. 1393
УДК 579.017.7; 620.95
СЫРОЙ ГЛИЦЕРИН КАК ПЕРСПЕКТИВНЫЙ СУБСТРАТ ДЛЯ ПРОИЗВОДСТВА ВОДОРОДА В ЛАТВИИ
И. Диманта1, А. Грудулс1, В. Николаева1, Я. Клеперис2, И. Муижниекс1
Латвийский университет, Факультет биологии Латвия, Рига, LV-1010, бульв. Кронвальда, д. 4 Тел.: 00 371 29776085, e-mail: ilze.dimanta@lu.lv 2Институт физики твердого тела при Латвийском университете Латвия, Рига, LV-1063, ул. Кенгарага, д. 8 E-mail: kleperis@latnet.lv
Заключение совета рецензентов: 20.08.12 Заключение совета экспертов: 25.08.12 Принято к публикации: 30.08.12
Производство водорода бактериями при ферментации - перспективный и привлекательный метод, т.к. в качестве субстрата могут использоваться различные возобновляемые ресурсы. Отходы производства биодизеля, а именно сырой глицерин, могут эффективно использоваться в производстве водорода; в Латвии доступны большие объемы этого сырья. Была изучена скорость производства водорода несколькими видами бактерий в разных тестовых установках. Было установлено, что несколько штаммов бактерий отвечают требованиям производства биоводорода из сырого глицерина.
Ключевые слова: биоводород, ферментация, сырой глицерин.
CRUDE GLYCEROL AS A PERSPECTIVE SUBSTRATE FOR BIO-HYDROGEN PRODUCTION IN LATVIA
I.Dimanta1, A.Gruduls1, V.Nikolajeva1, J.Kleperis2,I.Muiznieks1
'Faculty of Biology, University of Latvia 4 Kronvalda bulv., Riga, LV-1010, Latvia Tel.: 00 371 29776085, e-mail: ilze.dimanta@lu.lv 2Institute of Solid State Physics, University of Latvia 8 Kengaraga str., Riga, LV-1063, Latvia E-mail: kleperis@latnet.lv
Referred: 20.08.12 Expertise: 25.08.12 Accepted: 30.08.12
Hydrogen production via bacterial fermentation is perspective and environmentally viable because widely available renewable resources can be used as substrates. Biodisel production waste product, namely, crude glycerol can be effectively used for hydrogen production and large quantities of available crude glycerol are available in Latvia. Various bacterial isolates were tested for hydrogen gas production rates from glycerol with different test-systems. It was concluded that several of the isolated bacterial strains are suitable for bio-hydrogen production using crude glycerol as substrate.
Keywords: bio-hydrogen, fermentation, crude glycerol.
Organization(s): MSc.biol., currently PhD student in University of Latvia Faculty of biology. Education: Master's degree University of Latvia, Faculty of Biology (2009-2011), bachelor's degree University of Latvia, Faculty of Biology (2006-2009).
Experience: Institute of Solid State Physics, University of Latvia, Engineer (2008-currently). Main range of scientific interests: Biohydrogen, Alternative energies, Biotechnology. Publications: A bacterial hydrogen production test system for measuring H2 concentrations in liquids and gases. I. Klepere, I. Muiznieks, J. Kleperis. Latvian Journal of Physics and Technical Sciences 2 (2010) 60-68.
Ilze Dimanta
o International Scientific Journal for Alternative Energy and Ecology № 09 (113) 2012
28 © Scientific Technical Centre «TATA», 2012
Introduction
Biological processes of hydrogen recovery and collection from organic waste material resources facilitate recycling of sewage and are environmentally benign [1]. Prevalent of bacterial hydrogen generation processes, using the dark fermentation, are driven by the anaerobic metabolism of pyruvate, which is formed during the substrate catabolism. The pyruvate breakdown is catalyzed by one of two enzyme systems. Thus in both biological systems, the pyruvate generated by glycolysis is used to anaerobically produce acetyl CoA, from which ATP can be derived, and either formate or reduced ferredoxin - from which hydrogen can be derived [2]. Bacterial hydrogen production by fermentation of carbohydrate-containing substrates (organic industrial waste materials) is frequently preferred to photolysis, because it does not rely on the availability of light sources [3]. During the conversion of organic wastes, in anaerobic environment, hydrogen gas is produced as a by-product. Substantial factors like availability and cost are highly important in the selection of waste materials to be used in hydrogen production with fermentative bacteria [4].
Such industrial organic waste product as crude glycerol are perspective for usage in feedstock for hydrogen producing bacteria. The process of biodiesel production results in 41% (w/w) of crude glycerol [5]. Numerous anaerobic and facultatively anaerobic bacteria, e.g. belonging to family Clostridiaceae, Enterobacteriaceae and others, can produce hydrogen from carbohydrate containing organic wastes. Glycerol like glucose can be fermented by E. Coli to produce hydrogen at acid pH [6].
Substrate fermentation dynamics is highly affected by physical and chemical factors, for example, pH in the media has a substantial impact on fermentation end-product formation [7]. Environmental conditions like medium pH and temperature are the major parameters to be controlled in the hydrogen production, because they affect bacterial produced gas qualitative and quantitative content and hydrogen yield and rate. Hydrogen production using glycerol is 1.5-fold higher at pH 5.5 than at pH 6.5 [8].
Parameters of hydrogen gas are measured by classic volumetric, mass-spectrometric and chromatography methods or using chemical gas sensors. To measure hydrogen gas concentration in liquid a hydrogen electrode is used (Pt or other noble metal - gold, rhodium, palladium, etc.), for example, Clark electrodes, where the cathode is polarized versus internal Ag/AgCl anode which is placed behind an electrically insulating silicone rubber membrane [9-11].
Experimental Methods 1. Strains
Aneurinibacillus aneurinilyticus Microbial Strain Collection of Latvia (MSCl) 1018, Clostridium sporogenes MSCL 764, Eubacterium limosum MSCL
840, Kluyvera ascorbata MSCL 732, Paenibacillus pabuli MSCL 1006, isolated in Latvia, Escherichia coli BW25113 hyaB hybC hycA fdoG frdC ldhA aceE::kan (from prof. T.K. Wood, USA).
2. Media and experimental set up Bacterial cultures were inoculated in 200 ml flasks containing Luria-Bertani (LB) [5 g/L yeast extract, 10 g/L tryptone, 10 g/L sodium chloride, 15 g/L Bacto agar] [12]. The flasks were aerobically shaken at 37 °C for 12 hours at 150 rpm using a multi-shaker PSU-20 (BioSan, Latvia) with the exception of Clostridium sporogenes (cultivated in the Thioglycollate resazurin broth (BioRad, France) for two days without shaking). Optical density (OD) calibration curve was used to find out number of cells in 1mL of culture [13]. The bacteria concentration in the fresh culture was 107 CFU/mL. The overnight culture in LB liquid medium was mixed (1:1) with phosphate buffered saline (PBS) pH 7.3 (0.8 g/l NaCl, 0.2 g/l KCl, 1.43 g/L Na2HPO4, 0.2 g/L KH2PO4) in a vessel sterilized for measurements. The PBS contained a complex trace element medium pH 5.5 [0.039g/L Fe(NH4)2-SO4-6H2O, 0.172 mg/L Na2SeOs, 0.02 mg/L NiCl2, 0.4 mg/L (NH4)6MovO24] [14]. Crude glycerol (98% wt/wt, determined with HPLC analysis) from biodisel fuel production was used as substrate, final concentration of glycerol used was 240 mM. Glycerol was sterilized through 0.2 ^m membrane filters. Substrate was added at the start of experiment. Argon gas bubbling through the media was used to sustain anaerobic environment.
3. Analytical methods 3.1. Liquid phase analysis Experimental test-system with separate glass chambers for simultaneous measurements and hydrogen and oxygen Clark-type microsensors (Unisense, Denmark) were used to determine hydrogen production rate in the liquid phase. Both hydrogen and oxygen microsensors were connected with the signal amplifier - a pico-ammeter and an A/D current converter connected to PC using USB port. Microsensors were calibrated in liquid culture medium for zero concentrations. Sensors were calibrated in LB medium using 99.999% pure hydrogen and 99.99% pure argon. Microsensor tip was sterilized using 96% ethanol, 0.1 M NaOH and distilled water every time when it is taken out from the sample [15].
3.2. Gas analysis For hydrogen analysis in the gas phase the RGAPro-100 mass-spectrometer connected to the experimental test-system was used. Evolved gases were collected with the syringe from the tests system with graduated gas holder and tested qualitatively in the mass-spectrometer. Influence of partial pressure of hydrogen (pH2) was investigated by cultivating the strains in experimental bottles with various liquid to gas ratios.
Международный научный журнал «Альтернативная энергетика и экология» № 09 (113) 2012 © Научно-технический центр «TATA», 2012
Водородная экономика. Методы получения водорода
Results and Discussion
Three facultative anaerobic bacteria strains (Aneurinibacillus aneurinilyticus MSCL 1018, Kluyvera ascorbata MSCL 732, Paenibacillus pabuli MSCL 1006), one strictly anaerobic bacterium strain (Clostridium sporogenes MSCL 764) and one modified strain (E. coli BW25113 hyaB hybC hycA fdoG frdC ldhA aceE::kan) capability to produce hydrogen during fermentation of glycerol as substrate was investigated in different experimental systems. Bacterial strain hydrogen production rates using different substrate fermentation are shown in Table.
Hydrogen production measurements in liquid phase on the sample with different bacterial strains using glycerol as substrate are shown in Fig. 1. H2 production rate with C.sporogenes in liquid phase reached 1.5 mmol H2/L/h, in gaseous phase - 1.42 mmol H2/L/h. Hydrogen production rate is time dependent - higher rate of hydrogen production is at the fermentation process beginning when concentration increases, but after three hours of fermentation, it decreases. Measurements with A. aneurinilyticus, E. limosum, K. ascorbata, P. Pabuli and E.coli showed that these strains weren't producing
substantial amount of hydrogen using crude glycerol as substrate, for example, E.coli BW25113 produced 0.125 mmol/L/h and E.limosum produced 0.07 mmol H2/L/h. However, using 1M crude glycerol, E.coli BW25113 produced maximally 0.8 mmol H2/L/h in gaseous phase (measurements were made in prototype bioreactor).
Максимальные скорости производства водорода для всех культур и субстратов H2 production maximum rates with all strains and substrates
Bacterial strain H2 production rate, mmol/L/h
liquid phase gaseous phase
A. aneurinilyticus MSCL 1018 0.06 0
E. coli BW25113 0.125 0.04
K. ascorbata MSCL 732 0.09 0.04
C. sporogenes MSCL 764 1.5 1.42
E. limosum MSCL 840 0.07 0.18
P. pabuli MSCL 1006 0.08 0
Рис. 1. Измерение производства H2 в жидкой фазе для различных бактериальных культур на глицерине в качестве субстрата Fig. 1. Hydrogen production measurements in liquid phase on the sample with different bacterial strains using glycerol as substrate
Mass-spectrometric analysis was used to estimate hydrogen production generation results in gaseous phase as well as constancy of anaerobic conditions in the bioreactor test-system. The constancy of oxygen, nitrogen (not above 2% of total gas volume) gas concentrations evidences that the system had reliable anaerobic conditions (Fig. 2).
Рис. 2. Масс-спектроскопический анализ образца бактерий
E.coli BW25113 hyaB hybC hycA fdoG frdC ldhA aceE Fig. 2. Mass-spectrometric analysis of the sample with bacteria E.coli BW25113 hyaB hybC hycA fdoG frdC ldhA aceE
International Scientific Journal for Alternative Energy and Ecology № 09 (113) 2012
© Scientific Technical Centre «TATA», 2012
Results with the prototype bioreactor showed that medium stirring and gas sparging has a significant impact on hydrogen production efficiency. There was an experiment carried out with discontinuous stirring, as well as discontinuous gas sparging to explore their influence on hydrogen production. Constant stirring do increase the overall hydrogen concentration and when it is turned off, the hydrogen concentration slowly decreases in overall fermentation process. In gaseous phase concentration alterations are not very obvious until the saturation is reached. However, substantial increase in hydrogen concentration in gaseous phase is achieved when inert gas sparging is maintained periodically.
Conclusions
Developed methods and lab-scale test systems allow adequate substrate and bacterial strain hydrogen productivity estimation. Various bacterial strains were analyzed for hydrogen generation productivity parameters using glycerol as substrate. Two of the studied bacterial strains (Clostridium sporogenes MSCL 764 and Escherichia coli BW25113, respectively) showed comparatively high results and are good candidates for further investigations of bio-hydrogen production from crude glycerol, which is a perspective substrate to be used in fermentation process: rapidly convertible, large quantities of crude glycerol available in Latvia.
Acknowledgements
Authors acknowledge the support of Latvian National Research Programme Research Program in Energetics, prof. T.K. Wood (USA) for E. coli BW25113, engineer Laimonis Jekabsons for technical assistance, and the European Social Fund for the scholarship to Ilze Dimanta.
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