HOLLOW-FIBER BIOREACTOR FOR GLYCERIN CONVERSION INTO H2 BY BACTERIUM ENTEROBACTER AEROGENES Текст научной статьи по специальности «Промышленные биотехнологии»

ЭНЕРГИЯ БИОМАССЫ

ENERGY OF BIOMASS

Статья поступила в редакцию 21.06.10. Ред. рег. № 829 The article has entered in publishing office 21.06.10. Ed. reg. No. 829

УДК 579.6

БИОРЕАКТОР С ПОЛЫМИ ВОЛОКНАМИ ДЛЯ КОНВЕРСИИ ГЛИЦЕРИНА В H2 БАКТЕРИЯМИ ENTEROBACTER AEROGENES

С.А. Марков, Б. Валдрон

Государственный университет им. Остина Пи, кафедра биологии а/я 4718, Кларксвилл, штат Теннесси, 37044, США, Тел.: 1 (931) 221-7440, факс: 1 (931) 221-6323, e-mail: markovs@apsu.edu

Заключение совета рецензентов: 09.07.10 Заключение совета экспертов: 19.07.10 Принято к публикации: 29.07.10

В статье обобщены результаты исследования по превращению глицерина бактериями Enterobacter aerogenes в молекулярный водород (H2). Самые высокие скорости выделения H2 и этанола наблюдались при 2% содержании глицерина в минеральной среде роста бактерии. Лабораторный биореактор с полыми волокнами (объемом 35 мл) был сконструирован для продуцирования H2 и спирта (этанола) иммобилизованными клетками бактерии E. aerogenes. Эффективность поглощения глицерина бактериями составляла 86% за 3 дня работы биореактора и была выше эффективности поглощения глицерина бактериями в колбах за тот же период времени. Выделение H2 и спирта наблюдалось в биореакторе в течение 3 дней. Максимальное выделение водорода в биореакторе было 30 мл в час, а максимальная концентрация спирта составляла 10 мМ. Выход H2 в биореакторе составлял 0,77 (теоретический выход H2: 1 моль H2 на 1 моль глицерина). Водород из биореактора был использован в небольшом топливном элементе, который генерировал достаточно электричества для работы карманного вентилятора.

Ключевые слова: биоводород, глицерин, этанол, Enterobacter aerogenes, биореактор.

HOLLOW-FIBER BIOREACTOR FOR GLYCERIN CONVERSION INTO H2 BY BACTERIUM ENTEROBACTER AEROGENES

S.A. Markov and B. Waldron

Biology Department, SSC A225, Austin Peay State University P.O. Box 4718, Clarksville, TN 37041, USA Phone: 1 (931) 221-7440, Fax: 1 (931) 221-6323, e-mail: markovs@apsu.edu

Referred: 09.07.10 Expertise: 19.07.10 Accepted: 29.07.10

Hollow-fiber bioreactor for glycerin conversion into H2 by bacterium Enterobacter aerogenes. S.A. Markov and Barbara Waldron, Austin Peay State University, Department of Biology. The article surveys experimental data on conversion of glycerin into molecular hydrogen (H2) by bacterium Enterobacter aerogenes. The highest H2 and ethanol production rates were observed under 2% glycerin in the mineral growth medium. A lab-scale hollow-fiber bioreactor for conversion of glycerin into H2 and ethanol by immobilized E. aerogenes cells was constructed. The glycerin uptake efficiency by bacteria in the bioreactor was found to be 86% for 3 days and higher compared to the test tube experiment (60%). Hydrogen and ethanol production in the hollow-fiber bioreactor by E. aerogenes from glycerin (2% glycerin, v/v) was observed for 3 days. The maximal H2 production rate in a bioreactor was 30 mL per hour and the maximal achieved ethanol concentration was 10 mM. The yield of H2 from glycerin (0.77 mol/mol) was relatively high in the bioreactor for the first 3 days of bacterial cultivation. Theoretical yield is 1 mol of H2 produced per 1 mol of glycerin consumed. Hydrogen from the hollow-fiber bioreactor was directly injected into a small mV fuel cell (PEM fuel cell) and was capable to generate enough electricity to power a small motor (fan).

Keywords: biohydrogen, glycerin, ethanol, Enterobacter aerogenes, bioreactor.

Introduction

Glycerin is widely used in medical, food and pharmaceutical preparations. It has recently become an abundant commodity due to its generation as a byproduct of biodiesel production [1]. About one pound of

glycerin is created for every 10 pounds of produced biodiesel. World biodiesel production is predicted to reach 12 billion liters by 2010. Currently, a surplus glycerin is destroyed by incineration. However, incineration has serious environmental impact, which includes the production of greenhouse gases. Burning

International Scientific Journal for Alternative Energy and Ecology № 8 (88) 2010

© Scientific Technical Centre «TATA», 2010

glycerin produces nitrogen oxide as well as carbon dioxide (CO2), the primary greenhouse gas. The overproduction of glycerin considerably affects economic viability of the biodiesel industry. However, using the surplus of glycerin for biofuel generation such as biohydrogen (H2) or ethanol offers a number of considerable benefits for people, prosperity and our planet. Molecular hydrogen is an ideal fuel to be used for transportation [2]. Energy content of hydrogen is three times greater than in gasoline and four times higher than in ethanol. The hydrogen power was running NASA's space rockets for many years. Today, a growing number of automobile manufacturers around the world are making hydrogen-powered vehicles. Because of depleting supplies and the growing demand for oil, the use of H2 as a fuel can become a perfect alternative to gasoline. Our current fossil-fuel based economy generates CO2 which is causing global warming the planet. Switching from fossil fuels to H2 will eventually stop the buildup of greenhouse gases that trigger global warming. Hydrogen is a climate perfect fuel. It is called "the clean fuel of the future". Using H2 as the energy source produces only water, and it is also a renewable fuel, since H2 can be made from water again. The conventional industrial methods for H2 production are costly and the problem has been so far is to find a cheaper way to produce H2. The biological H2 production by microbial species has a number of advantages, and it can be a cost effective alternative to the current industrial methods of H2 production. Ethanol is also an excellent transportation fuel that is used as a gasoline substitute. In respect to its environmental attributes, it is superior to gasoline when produced from non-crop biomass. Compared to gasoline, ethanol burns more cleanly. Ethanol-fueled vehicles produce lower CO and CO2 emissions. It is considerably less toxic to humans than gasoline.

There are few bacteria that are able to convert glycerin into H2 and ethanol. These are the strains of Klebsiella, Citrobacter, Clostridium, Enterobacter, and Escherichia coli [3, 4]. Most importantly, some bacteria such as Enterobacter ferment glycerin under anaerobic conditions into H2 and ethanol with a minimal amount of other by-products [3]. Bacteria cannot grow on pure glycerin; it must be diluted in nutrient medium. Thus, to minimize the cost of biofuel generation from glycerin and improve its efficiency, it is desirable to increase cell to glycerin contact surface as high as possible. A specific bioreactor design is required to allow mass transport of glycerin to cells. In this study, we used a simple hollow-fiber bioreactor to increase mass transfer of glycerin to bacterial cells. Hollow fibers are made of semipermeable polymeric membranes. Salts and gases can freely diffuse through these membranes. Bacterial cells, due to their larger size, cannot pass through the membrane. Bacterial cells that grow on hollow fibers are called immobilized cells. The hollow-fiber bioreactor usually includes hundreds of hollow fibers with immobilized cells on them [5]. Large surface-to-volume ratio of the hollow

fibers will allow us to grow bacterial cells in bioreactors in high densities, and to promote mass transport of glycerin to cells. Another advantage is that biorectors with immobilized on hollow-fibers bacteria can be operated continuously over a long period of time, more than one year [6].

Materials and methods

Bacterial culture Prior to the inoculation of bacterial cells into bioreactors the bacterium, Enterobacter aerogenes from Carolina Biological Supply Company, was grown aerobically in test tubes (5.0 mL suspension volume) on the synthetic medium containing inorganic salts and glycerin [3]. Cell dry weight was determined by trapping the bacteria on Whatman filter paper and drying the cell suspensions at 90 °C to a constant weight.

Bioreactor

A lab-scale hollow-fiber bioreactor was constructed (Fig. 1). A 153 mm x 33 mm AM-06 cartridge with hydrophilic cuprammonium rayon hollow fibers (Asahi Kasei Medical CO Ltd, Japan) was used as the bioreactor column. The total surface area of the hollow fibers was 0.6 m2 and the cartridge volume outside of the fibers was 35 mL. The diameter of each hollow-fiber was 180 ^m. The bioreactor was designed in a way that the glycerin was diluted in the growth medium, and then was added using a LKB 2232 MicroPerpex peristaltic pump (Pharmacia LKB Biotechnology, Sweden) from the outside of the fibers within the column. Bacterial cells were readily adsorbed to the outer surface of the hollow fibers and consumed the glycerin. The used growth medium went into hollow fiber lumen and returned to the bioreactor medium reservoir, in order to create a closed system in which it was possible to measure glycerin uptake and ethanol production by the cells. The bioreactor was functioning under 37 °C.

/_ medium flow

pump

cjiii il' rlllllllDl

111 ГНК Mini llM'f

Рис. 1. Схематическая диаграмма биореактора с полыми волокнами для непрерывного выделения молекулярного водорода из глицерина иммобилизованными бактериями E. aerogenes

Fig. 1. Schematic representation of a hollow-fiber bioreactor for continuous production of H2 and ethanol from glycerin by immobilized E. aerogenes

Международный научный журнал «Альтернативная энергетика и экология» № 8 (88) 2010 © Научно-технический центр «TATA», 2010

The bioreactor was inoculated with 5 mL of bacterial seed culture at cell density of 0.65 g/L. Before inoculation the bioreactor was sterilized with 5% bleach solution and with subsequent cleaning using sterile distilled water.

the initial and final concentrations of glycerin, respectively. The glycerin uptake efficiency was 40% at an original concentration of 1%; 70% at an original concentration of 2%; and 60% at an original concentration of 10% in seven days.

Measurement of hydrogen production Hydrogen production was measured using a Gow-Mac gas chromatograph (Bethlehem, PA) equipped with a molecular sieve 5A column and a thermal conductivity detector. Nitrogen was used as the carrier gas.

Ethanol and glycerin measurements Ethanol and glycerin concentrations were determined spectrophotometrically using an ethanol or glycerin kits (BioVision, CA) and ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).

Results

Hydrogen and ethanol production in test tubes

1. Calculation of glycerin uptake efficiency Prior to construction of the bioreactor, we investigated the conditions for maximal H2 production by batch cultures in tubes, to test the system that is less complex than a bioreactor. First, we studied if cells of E. aerogenes were able to grow on a synthetic medium with glycerin. In these experiments, different amounts of glycerin (v/v) were added to the medium. Bacterial cells grew and consumed glycerin better at an original concentration of 1% or 2%. No significant growth was observed on a synthetic medium with 0.1% glycerin. The consumption of glycerin by bacteria is shown on Table 1.

Таблица 1

Поглощение глицерина бактериями E. aerogenes при различных концентрациях глицерина (1%, 2% и 10% в синтетической среде)

Table 1

Glycerin consumption by batch cultures E. aerogenes under different initial concentration of glycerin (1%, 2% and 10% in synthetic medium) for a seven day period

Day Glycerin concentration, % Initial concentration

1% 2% 10%

0 1.00 2.00 10

1 0.85 1.00 7.3

2 0.75 0.90 5.2

3 0.60 0.80 3.3

6 0.60 0.70 3.0

7 0.60 0.60 2.8

2. Hydrogen and ethanol production rates Hydrogen and ethanol production were measured under different concentration of glycerin in the synthetic medium. The highest H2 and ethanol production rates were observed under 2% glycerin (v/v) in the synthetic medium (Fig. 2). Ethanol data is not shown.

700

500

! * <p "a

300

100

0 2 4 6 8 10 12

Glycerin (% v/v in the medium)

Рис. 2. Выделение водорода однодневной культурой E. aerogenes как функция концентрации глицерина в среде роста Fig. 2. Hydrogen production by one day-old culture of E. aerogenes as a function of glycerin concentration in the growth medium

3. Hydrogen and ethanol production under optimal

glycerin conditions After determination of the optimal concentration of glycerin in the synthetic medium for H2 and ethanol production, we studied H2 and ethanol generation by bacteria during a several day period under 2% and 4% glycerin (v/v) in the medium (Table 2). Hydrogen production was highest during the first day of running the batch culture, but ethanol production was highest at the later days.

Таблица 2

Выделение водорода и этанола E. aerogenes в колбах в зависимости от времени эксперимента

Table 2

Hydrogen and ethanol production by E. aerogenes in test tubes as a function of time

The glycerin uptake efficiency by E. aerogenes was calculated. The glycerin uptake efficiency (E) was defined as: E = [(/ -F)/I] x 100%, in which I and F are

Days H2 production, mL-g"1 dry weight-h"1 under glycerin Ethanol production, mM under glycerin

2% 4% 2% 4%

1 628 42 0.65 0

2 49 46 1.8 1.4

4 32 9 5 2.7

International Scientific Journal for Alternative Energy and Ecology № 8 (88) 2010

© Scientific Technical Centre «TATA», 2010

Hydrogen and ethanol production in a hollow-fiber bioreactor

1. Calculation of glycerin uptake efficiency The glycerin uptake efficiency in a bioreactor was calculated using a formula as shown above. The efficiency of glycerin uptake was 86% in 3 days (glycerin content in the bioreactor medium decreased by 86% in 3 days) compared to 60% in batch cultures for the same time period (Table 1).

2. Hydrogen and ethanol production The assessment of H2 and ethanol-production activity by bacteria in the hollow-fiber bioreactor in the presence of 2% glycerin (v/v) was made. Hydrogen production (Fig. 3) in the hollow-fiber bioreactor by E. aerogenes from glycerin was observed for three days at the maximum rate of 30 mL per hour. Hydrogen from the hollow-fiber bioreactor was directly injected into a small mV fuel cell (PEM fuel cell) and was capable to generate enough electricity to power a small motor (fan).

Il [}..ri 1 1.5 f Л1. л

Days

Рис. 3. Продукция водорода бактерией E. aerogenes в биореакторе с полыми волокнами Fig. 3. Hydrogen production by E. aerogenes in the hollow-fiber bioreactor

Together with H2, the bacterial cells produced ethanol (Fig. 4). Both H2 and ethanol production by E. aerogenes in the hollow-fiber bioreactor was higher compared to H2 and ethanol production in the test tubes.

Рис. 4. Продукция спирта бактерией E. aerogenes в биореакторе с полыми волокнами Fig. 4. Ethanol production by E. aerogenes in the hollow-fiber bioreactor

3. Yields of hydrogen and ethanol from glycerin The yield of H2 from glycerin (2% v/v) on the 3rd day of bacterial growth was relatively high at 0.77 mol of H2 per mol of glycerin. The ethanol yield was 0.05 mol/mol of glycerin for the same time period.

4. Energy conversion efficiency of glycerin into hydrogen The energy conversion efficiency of glycerin (2% glycerin v/v) into H2 by E. aerogenes was calculated. The heat of H2O formation, (241 KJ-mol-1) was used as the energy content of the H2 produced. The heat of combustion of glycerin is 1571 KJ-mol-1. The yield of H2 from glycerin is 0.77 mol/mol of glycerin as shown above.

Energy conversion efficiency: (241x0.77)/1571x100% = 12%

Discussion

In this study the hollow-fiber bioreactor was tested for H2 and ethanol production by bacterium E. aerogenes from glycerin. We exceeded our expectations by obtaining higher H2 production rates (Table 3) from glycerin compared to H2 rates from other microorganisms reported in published papers [7-9].

Таблица 3

Скорости продукции молекулярного водорода микроорганизмами из литературных источников и наших экспериментов

Table 3

Rates of microbial H2 production obtained from literature and from our experiments

Microorganism Rate of hydrogen production (mL-g-1 dry weight-h"1)

Anabaena variabilis via photosynthèses [7] 1G

Rubrivivax gelatinosus via gas-shift reaction [8] 125

Enterobacter aerogenes via fermentation of sugar [9] 4GG

E. aerogenes via fermentation of glycerin [our results] 62 S

That was possible by choosing the right bacterium with high yields of H2 production from glycerin, and by increasing mass transfer of glycerin into the bacterial cells using hollow fibers in the bioreactor. The large surface-to-volume ratio of the hollow fibers allows to grow cell cultures in high densities in bioreactors, and to promote mass transport of glycerin to cells. Membrane bioreactors (such as our hollow-fiber membrane bioreactor) are a technology which is actively being pursued recently in water purification [10]. The glycerin uptake efficiency was higher in our bioreactor (86%)

Международный научный журнал «Альтернативная энергетика и экология» № 8 (88) 2010 © Научно-технический центр «TATA», 2010

compared to the batch culture experiment (60%). Our bioprocess for conversion of glycerin into H2 is ready for practical application. The H2 from a hollow-fiber bioreactor is clean for direct injection into a H2 fuel cell. In fact, it has been directly injected into small mV fuel cells and was shown capable of generating enough electricity to power a small motor. No negative effect on the fuel cells was noted.

Conclusions

1. The results presented here demonstrate that there is a potential for the use of hollow fiber-immobilized bacterial cells in a bioreactor for H2 production from glycerin and to use this H2 in fuel cells to generate electricity.

2. The large surface-to-volume ratio of the hollow fibers allows to grow bacterial cells in high densities in bioreactors and to design compact systems.

Acknowledgements

This work was supported by the National Science Foundation (USA) and the U.S. Environmental Protection Agency P3 Program. The authors thank Asahi Kasei Medical CO Ltd (Japan) for supplying the hollow fiber cartridges.

References

1. Yazdani S.S., Gonzales R. Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products // Metabolic Engineering. 2008. Vol. 10. P. 340-351.

2. Cammack R., Frey M., Robson R. L. Hydrogen as Fuel: Learning from Nature. London: Taylor & Francis, 2001.

3. Ito T., Nakashimada Y., Senba K., Matsui T., Nishio N. Hydrogen and ethanol production from glycerol-containing wastes after biodiesel manufacturing process // Journal of Bioscience and Bioengineering. 2005. Vol. 100. P. 260-265.

4. Yazdani S.S., Gonzalez R. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry // Current Opinion in Biotechnology. 2007. Vol. 18. P. 213-218.

5. Bunch A.W. The uses and future potential of microbial hollow-fibre bioreactors// J. Microbiol. Methods. 1988. Vol. 8. P. 103-119.

6. Markov S.A. Bioreactors for hydrogen production // O.R.Zaborsky (ed.) Biohydrogen, 1998. P. 393-390.

7. Tamagnini P., Axelsson R., Lindberg P., Oxelfelt F., Wunschiers R., Lindblad P. Hydrogenases and hydrogen metabolism of cyanobacteria // Microbiology and Molecular Biology Review. 2002. Vol. 66, №. 1. P. 1-20.

8. Markov S.A., Weaver P.F. Bioreactors for H2 production by purple nonsulfur bacteria // Appl. Biochem. Biotecnol. 2008. Vol. 145. P. 79-86.

9. Tanisho S. Feasibility study of biological hydrogen production from sugar cane by fermentation // Hydrogen Energy Progress XI. Proc. 11th World Hydrogen Energy Conference, Stuttgart, Germany. 23-28 June 1996. 1996. Vol. 3. P. 2601-2606.

10. Shannon M.A., Bohn P.W., Elimelech M., Georgiadis J.G., Marinas B.J., Mayes A.M. Science and technology for water purification in the coming decades // Nature. 2008. Vol. 452. P. 301-310.

International Scientific Journal for Alternative Energy and Ecology № 8 (88) 2010

© Scientific Technical Centre «TATA», 2010