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11Статья поступила в редакцию 07.08.12. Ред. рег. № 1394 The article has entered in publishing office 07.08.12. Ed. reg. No. 1394
УДК 579.2; 620.95; 628.336.6
ДИЗАЙН ПРОСТОГО БИОРЕАКТОРА ДЛЯ ПРОИЗВОДСТВА ВОДОРОДА И МЕТАНА БАКТЕРИЯМИ -ОПТИМИЗАЦИЯ И ЭКСПЕРИМЕНТЫ
12 12 1 1 2 А. Грудулс', И. Диманта', И. Дирнена , И. Муижниекс , Я. Клеперис
'Латвийский университет, Факультет биологии Латвия, Рига, LV-1010, бульв. Кронвальда, д. 4 E-mail: arturs.gruduls@gmail.com, ilze.dimanta@lu.lv, www.lu.lv 2Институт физики твердого тела при Латвийском университете Латвия, Рига, LV-1063, ул. Кенгарага, д. 8 E-mail: kleperis@latnet.lv, www.cfi.lv
Заключение совета рецензентов: 20.08.12 Заключение совета экспертов: 25.08.12 Принято к публикации: 30.08.12
Получение водорода путем реакций ферментации - это сложный процесс, на который влияют различные физические, химические и биологические факторы, а также тип реактора. В экспериментах использовались различные штаммы E. coli (дикий 332 и измененный BW25113 hyaB hybC hycA fdoG frdC ldhA aceE). Наша лабораторная тестовая система состоит из 2 реакторов: в первом производится биоводород, во втором - биогаз (метан). За счет подачи неочищенного биоводорода из первого реактора мы ожидаем увеличения производства метана во втором. Результаты экспериментов показали, что большое влияние на эффективность произодства водорода оказывают помешивание и барботаж. Для дальнейших экспериментов необходим специальный тестовый стенд.
Ключевые слова: биореактор, ферментация, биогаз.
SIMPLE BIOREACTOR DESIGN FOR HYDROGEN AND METHANE GAS PRODUCING MICROORGANISMS - OPTIMIZATION AND EXPERIMENTS
12 12 1 1 2 A. Gruduls ' , I. Dimanta, ' , I. Dirnena , I. Muiznieks , J. Kleperis
'Faculty of Biology, University of Latvia 4 Kronvalda bulv., Riga, LV-1010, Latvia E-mail: arturs.gruduls@gmail.com, ilze.dimanta@lu.lv, www.lu.lv 2Institute of Solid State Physics, University of Latvia 8 Kengaraga str., Riga, LV-1063, Latvia E-mail: kleperis@latnet.lv, www.cfi.lv
Referred: 20.08.12 Expertise: 25.08.12 Accepted: 30.08.12
Hydrogen production using bacterial fermentation process is a very complex process and is influenced by many physical, chemical and biological factors, as wel as, by fermentation reactor type. Wild type E. coli 332 and modified E. coli BW25113 hyaB hybC hycA fdoG frdC ldhA aceE strains were used for biohydrogen production experiments. Constructed laboratory scale bioreactor system consists of two reactors. First reactor is for biohydrogen production and second for biogas (methane) production. Our expectation was that delivering unrefined biohydrogen gas from first reactor into biogas reactor will result in CH4 outcome increase. Experimental results showed that stirring and gas sparging has significant impact on hydrogen production efficiency. Further experiments require specific laboratory scale test-systems.
Keywords: bioreactor, fermentation, biogas.
International Scientific Journal for Alternative Energy and Ecology № 09 (113) 2012
© Scientific Technical Centre «TATA», 2012
Arturs Gruduls
Organization(s): MSc student at Faculty of Biology, University of Latvia, and Engineer in Institute of Solid State Physics, University of Latvia.
Education: Bachelor degree from Faculty of Biology, University of Latvia (2008-2011).
Experience: Engineer (2008-2012) at Institute of Solid State Physics University of Latvia; participant at 2 Research projects and National Research Program.
Main range of scientific interests: Hydrogen technologies, biohydrogen, biogas technologies, microbial fuel cells.
Publication: A. Gruduls, I.Dirnena, I. Klepere, I. Muiznieks, J. Kleperis. "Building up bioreactor prototype and optimisation for experiments with hydrogen and methane gas producing microorganisms" Paper No 320BI0; 1th International Congress on Hydrogen Production ICH2P-11, Thessaloniki, Greece, June 19-22, 2011; Conference Abstracts Proceedings in CD form.
Introduction
As the world reserves of fossil fuels are limited and will be exhausted sooner or later, active research interest has been stimulated in nonpetroleum, renewable and nonpolluting fuels. Biogas production technologies are way ahead biohydrogen production. However hydrogen is a promising alternative to fossil fuels and is considered to be very efficient energy carrier of the future. Hydrogen has a high energy yield (122 kJ/g), which is about 2.75 times greater than that of most effective hydrocarbon fuels [1]. Fermentative hydrogen production can use various organic wastes as substrate for biohydrogen production. Hydrogen production from renewable organic waste represents an important area of green bioenergy production and bioremediation. Biohydrogen is produced during fermentation processes. Various combinations of bacteria strains and substrates are explored to obtain highest production rates. Fermentative hydrogen production is a very complex process and influenced by many physical, chemical and biological factors and also fermentation reactor type. Therefore the design and optimisation of industrial fermentation processes requires the experimental investigation in small scale laboratory test-systems [2]. However commercial laboratory test-systems and bioreactors vary a lot and are designed for many different scientific purposes, they are quite expensive and often have some disadvantages for specific experiments and are not multifunctional. Instead of buying a new appropriate commercial test-system each time we meet specific drawback with existing system we should consider using and developing cheap multifunctional test-systems that could be rapidly modified if needed.
Most of the studies on fermentative hydrogen production are conducted in batch type mode due to its simple operation and control [3, 4, 5]. In these studies mostly batch type reactors without stirring or inert gas sparging are used. In our work we have examined the influence of stirring and inert gas sparging on hydrogen production efficiency. 0ur first reactor test-system (Fig. 1) was made to find out effects of these two factors.
Рис. 1. Схематическая диаграмма сконструированной нами тестовой системы для анаэробной культивации Fig. 1. Schematic diagram of our first constructed anaerobic cultivation test system
Henri law equation Eq. (1) shows close connection between dissolved hydrogen concentration and partial pressure of hydrogen in gaseous state above the liquid, where p - partial pressure, kH - constant, c -concentration of dissolved gas [6]:
P = км c.
(1)
An inert gas heavier than hydrogen can be used to extract hydrogen from liquid phase to increase its concentration in gaseous phase. Experiment results showed that stirring and gas sparging has significant impact on hydrogen production efficiency and will be discussed later. Optimization of first system was carried out reflecting on results of various experiments, compatibility requirements with commercial sensors and test probes, reducing build-up and maintenance costs and future predictions on prototype reactor operation.
Международный научный журнал «Альтернативная энергетика и экология» № 09 (113) 2012 © Научно-технический центр «TATA», 2012
Our constructed laboratory scale bioreactor system consists of two reactors: one for biohydrogen production and second for biogas production. However each reactor can be used as separate unit. It is reported that hydrogen utilizing methanogens are critical factor for the anaerobic bio-hydrogen production due to the rapid H2 uptake during fermentation [7]. Therefore it could be possible to increase biogas outcome by adding extra bio-hydrogen to reactor. System is made to be hermetic and compatible with different size commercial sensor probes using a specially developed fast coupling system. Testsystem can be modified and can be used to analyse different fermentative processes using various microorganisms. System allows monitoring of hydrogen or oxygen concentration simultaneously in liquid phase and gaseous phase. It is possible to monitor gas composition with masspectrometer during fermentation processes. In further development system will be fully automated to provide full control over experiment's parameters like temperature, pH, periodic aeration, substrate and products concentrations etc. First of all new prototype reactor system will be tested to examine how to improve biogas production using extra biohydrogen. Furthermore experiments will be done to examine and improve biohydrogen production, purification, collection and storing. Biohydrogen and biogas will be produced from alternative local resources - wastes and byproducts of food industry probably also using local wild type bacteria strains. We expect that delivering unrefined biohydrogen gas from first reactor into biogas reactor will increase CH4 outcome.
Materials and methods
Two Escherichia coli (E. coli) strains were used for first test experiments E. coli 332 and modified E. coli BW25113 hyaB hybC hycA fdoGfrdC ldhA aceE that has up to 4.6 times increased hydrogen production capability [3]. Bacteria were aerobically cultivated in Luria Bertrani (LB) medium that contained 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl [8]. Cultivation was carried out in shake flasks for 12 hours at 37 °C using Multi-shaker PSU-20 at 120 rpm. Optical density (OD) calibration curve was used to find out number of cells in 1mL of culture [9]. It was assumed that each E. coli cell contains 1.5410-13 grams of protein [10]. In sterile testsystem 250 mL of culture was mixed with 250 mL of phosphate buffer. Buffer composition was 0.8 g/L NaCl, 0.2 g/L KCl, 1.43 g/L Na2HPO4, 0.2 g/L KH2PO4 [11]. Glucose was used as a model substrate to observe dynamic of hydrogen production in test-system. 2 mL of 50% glucose was added to culture in test-system. System was placed in thermostat at 37 °C.
Unisense A/S hydrogen microsensor "H210" was used for dissolved hydrogen detection in liquid phase. Measurements were started after glucose was added to culture. Sensor was calibrated in LB medium using 99.999% pure hydrogen and 99.99% pure argon gas.
Mass-spectrometer RGAPro 100 was used for hydrogen detection in gaseous phase. For each sample 20 cubic centimeters of gas were taken. There was 200 mL headspace for gas in the test-system. Concentration of hydrogen in gaseous phase was calculated using volumetric data from masspectrometer and simple calculations Eq. (2). End-result units were mmol/L.
M-ptn-000. (2)
Different materials were used to build up the prototype of bioreactor. Hardened glass vessels of different volumes (0.75 L - 3 L) were purchased together with top sealing lids from stainless steel. For stirring common 4.5V DC motors sealed in hermetic housing were used. Special porous, thermostable (up to 200 °C) silicon rubber was used for gaskets and as sealing material. Sensor probe holders, valves, gas inlets and outlets were made from Ertacetal H-TF because it is resistant to chemicals, mechanical degradation, easy to handle within machining process and is stable up to 175 °C (that is far enough for sterilization in autoclave at 120 °C).
Results and discussion
Importance of stirring and inert gas sparging
Hydrogen production efficiency for wild type E. coli 332 strain (Fig. 2) is lower than that of modified E. coli BW25113 hyaB hybC hycA fdoG frdC ldhA aceE (Fig. 3) strain. Maximal hydrogen concentration that was achieved using wild type strain was 1.32 mmol/L in liquid phase and 0.40 mmol/L in gaseous phase. However as for modified strain these concentrations went up to 4.0 mmol/L in liquid phase and 6.9 mmol/L in gaseous phase.
Stirring and inert gas sparging has significant effect on effectiveness of hydrogen production in lab scale test-systems (Fig. 3, Fig. 4). These results show that stirring and inert gas sparging increased hydrogen concentration in liquid phase from 4.0 mmol/L to 9.0 mmol/L and from 6.9 mmol/L to 8.1 mmol/L in gaseous phase. Maeda [3] concluded that, using modified E. coli BW25113 hyaB hybC hycA fdoG frdC ldhA aceE strain, highest achieved hydrogen production efficiency is 44 ^mol H2/mg of protein. Our achieved efficiency is 47 ^mol H2/mg of protein. The concentration alteration in liquid phase is greater than that in gaseous phase. This could be explained due to gradual hydrogen accumulation in headspace above the medium with culture. Thus the partial pressure of gaseous hydrogen increases. As showed in Henri equation (Eq. 1) increase of partial pressure in gaseous phase increases the concentration of dissolved gas. Produced hydrogen had no opportunity to exit system and it accumulated in liquid phase, thus probably slowing down entire production.
International Scientific Journal for Alternative Energy and Ecology № 09 (113) 2012
© Scientific Technical Centre «TATA», 2012
Е. с olí 332
roo 2.00 з.оо 4.00 5.0:0 е.оо т.оо
time (hours)
Рис. 2. Сравнение количества произведенного штаммом E. coli 332 Н2 в растворенном и газообразном видах в анаэробной системе при помешивании и барботаже аргоном Fig. 2. Comparison of H2 production by E. coli 332 strain in dissolved and gaseous phases in anaerobic system with stirring
and argon sparging
E.coli BW25113 hyaB hybC hycA fdoG frdC IdhA aceE
1.00 2,00 3.00 4.00 5.00 6.00 7.00 8.00 9 00
time (hours)
Рис. 3. Сравнение количества произведенного штаммом E. coli BW25113 Н2 в растворенном и газообразном видах в анаэробной системе без помешивания и барботажа аргоном Fig. 3. Comparison of H2 concentration produced by E. coli BW25113 in dissolved and gaseous phase. Anaerobic system without stirring and argon sparging
E.coli BW25113 hyaB hybC hycA fdoG frdC IdhA aceE
—H2 liquid phase -U-H2 gaseous phase Щ
Я 1 jjj 8,08
7.14 t
I - ~~piK
5,45
[|M 4,48
d 11 3,44
2,7 ,14 2,4
IrM:
ОД4 —™t П1В 0,36
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
time (hours)
Рис. 4. Сравнение количества произведенного штаммом E. coli BW25113 Н2 в растворенном и газообразном видах в анаэробной системе при помешивании и барботаже аргоном Fig. 4. Comparison of H2 concentration produced by E. coli BW25113 in dissolved and gaseous phase. Anaerobic system with stirring and argon sparging
Международный научный журнал «Альтернативная энергетика и экология» № 09 (113) 2012 © Научно-технический центр «TATA», 2012
£, coli BW25113 hyaB hybC hycA fdoC frdC idhA aceE
Ü.ÜO 1,00 2,00 3,00 4,00 5,00 6,00
timf> ( hours)
7.-J0
8 00
9.00
1 0,00
Рис. 5. Изменения в концентрации водорода в растворенном и газообразном видах в зависимости от помешивания и барботажа: Ar+ - барботаж аргоном; Ar- - без барботажа; S+ - помешивание; S- - без помешивания; H2 - концентрация водорода в газообразной фазе (объемные %) Fig. 5. Changes in hydrogen concentration in dissolved and gaseous state depending on sparging and stirring effects: Ar+ - sparging with argon; Ar- - sparging turned off; S+ - stirring on; S- - stirring off; H2 - hydrogen concentration in gaseous state (volume %)
An additional experiment was carried out with discontinuous stirring and discontinuous gas sparging (Fig. 5) to explore how each of these two factors influence hydrogen production effectiveness. Results showed that constant stirring increase overall hydrogen concentration in liquid phase. If stirring is stopped concentration decreases. Concentration alterations in gaseous phase are not very obvious until saturation is not reached. Fast increase in hydrogen concentration in gaseous phase can be achieved due to inert gas sparging thus decreasing concentration in liquid phase and promoting overall hydrogen fermentative production. However argon is quite expensive gas. Gas sparging should be used only periodically as hydrogen concentration in liquid phase has reached saturation. Thereby decreasing systems maintenance costs.
Optimized laboratory scale test-system
Large scale bioreactors have some drawbacks on even substrate diffusion due to their great volume [12]. Small scale-down test-systems could be used to simulate
and solve these problems [13]. Our new multifunctional bioreactor prototype schematic is shown in Fig. 6. It can be used as an adjustable laboratory scale test-system for various fermentation experiments. Medium inlet valve and culture outlet valve makes it possible to use this system also as a continuous flow hemostat.
With external automatics it is possible to control different parameters for fermentation process. Heating is provided with external thermostat. Central processing unit (CPU) processes incoming data from pH probe (Fig. 7) and automatically regulates pH of medium. When hydrogen concentration in liquid phase reaches saturation, CPU triggers gas inlet valve to open. Sparging with argon is the initiated until hydrogen concentration in medium is greatly reduced. This conformation of system is a typical application for simple fermentation experiments. Composition of headspace gas can be measured with masspectrometer. For quantitative analysis produced gas is bubbled trough sodium hydroxide to get rid of excess carbon dioxide. It is accumulated in special vessel.
International Scientific Journal for Alternative Energy and Ecology № 09 (113) 2012
© Scientific Technical Centre «TATA», 2012
Рис. 6. Схематическое изображение разрабатываемого прототипа одной ячейки-реактора нового биореактора Fig. 6. Schematic diagram of new bioreactor prototype single reactor unit under construction process
Рис. 7. Типичные применения водорода и эксперименты по получению метана Fig. 7. Typical application for hydrogen and methane production experiments
0ur constructed bioreactor system for biogas production experiments consists of two separate reactors (Fig. 8). First stage is used for biohydrogen production. Unpurified biohydrogen is then passed trough microbial filter to second stage reactor. In second stage methanogenic bacteria are used for biogas production. C02 sensor measures the intensity of fermentation while CH4 sensor measures concentration of methane in exhaust gas. More accurate results are achieved when masspectrometer is used. We expect that delivering unrefined biohydrogen gas from first reactor into biogas reactor will result in increase in CH4 outcome.
Рис. 8. Прототипы систем для получения водорода и более эффективное производство биогаза Fig. 8. Bioreactor system prototype for biohydrogen and more efficient biogas production
Международный научный журнал «Альтернативная энергетика и экология» № 09 (113) 2012 © Научно-технический центр «TATA», 2012
Conclusions
Hydrogen production due to fermentation processes is more efficient if continuous stirred tank reactor type system is used instead of simple batch-type tank reactor. A significant increase in hydrogen concentration in gaseous phase can be achieved due to system sparging with inert gas like argon or nitrogen.
In order to reduce costs for reactor maintenance sparging should be periodic not continuous, for example only than when concentration of hydrogen in dissolved state has reached saturation.
Small scale, low cost bioreactors could be successfully used in laboratories to simulate and solve problems of large scale commercial fermentation processes. Multi functional and fully adjustable testsystems have significant advantages instead of highly specialized units.
Acknowledgement
Authors Arturs Gruduls and Ilze Dirnena acknowledge the financial support of European Social Fund within the project "Support for Master Studies at University of Latvia" and Ilze Dimanta acknowledge the financial support of European Social Fund within the project "Support for Doctoral Studies at University of Latvia". Financial support of National Research Program in Energetics is gratefully acknowledged.
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International Scientific Journal for Alternative Energy and Ecology № 09 (113) 2012
© Scientific Technical Centre «TATA», 2012
