Hydrogen Production by Rhodobacter Sphaeroides O.U.001

Introduction (TOP)

Hydrogen is a clean and efficient fuel, and a potential substitute for fossil fuels in the long run. 

Biological hydrogen production is environmentally friendly, uses renewable resources and does not require complex equipment 

Currently low production rates and high substrate costs limit the economical feasibility of biological hydrogen production. Increasing feasibility requires detailed research. 

Objectives (TOP)

Studies made by our group aim the following: 

Development of an understanding of the hydrogen production characteristics of the bacteria 

Increasing the hydrogen production rates through optimization of variables 

Investigation of the possibilities of decreasing the cost 

Scaling-up of the photobioreactor for the biological hydrogen production.

Microorganisms Producing H₂ (TOP)

Algae (Oxygenic photosynthesis) 

 H2O à H2 + ½ O2

Cyanobacteria (Oxygenic photosynthesis) 

H2O à H2 + ½ O2

Anaerobic bacteria (Fermentation) 

 C6H12O6 à H2+ CO2+ organic acids

Photosynthetic bacteria (Anoxygenic photosynthesis) 

organic acids + H2O à H2 + CO2

Studies of the M.E.T.U. Biohydrogen group with R.sphaeroides OU 001(TOP)

Hydrogen Production Setup (TOP)

Typical Conditions (TOP)

The photosynthetic bacteria evolve hydrogen from organic carbon compounds when subjected to illumination under an inert anaerobic atmosphere. Below are typical conditions used in hydrogen production experiments. 

Temperature: 30-35 °C 

Initial pH: 6.8-7.0 

Nutrient medium: (Modified from Biebl and Pfennig, 1981) 

Malate / Wastewater (Carbon) Glutamate (Nitrogen) Salts+Vitamins 

Light Intensity: (Tungsten)150-250 W/m²

Atmosphere: Pure argon 

Volume: 400 ml

Selection of microorganism (TOP)

Each group of microorganism has its advantages and drawbacks for hydrogen production. The following advantages of Photosynthetic Bacteria make them particularly suitable for biological hydrogen production.

High substrate conversion efficiency.

Ability to utilise a wide wavelength range of sunlight.

Ability to utilise a large variety of compounds for growth or H2 production.

Ability to survive under changing conditions. Among the photosyntetic bacteria, R. sphaeroides was observed to have a high hydrogen production rate. Therefore this bacterium was selected and studied for hydrogen production.

Factors Affecting Hydrogen Production  (TOP)

Effect of temperature and light intensity 

There are many factors which influence hydrogen (Arik et. al.,1996) production by R. sphaeroides in one way or the other. First, the effect of parameters that could be easily manipulated and set constant- such as temperature, light intensity- were investigated. 

Table 1. Influence of temperature and light intensity

Effect of Substrate Concentrations 

Then the effect of substrate concentrations was examined (Eroğlu et. al.,1999). It was found that high concentrations of glutamate promoted growth at the expense of hydrogen production. The effect of glutamate concentration is seen in Figure 2. 

Figure 2 – Hydrogen produced with 15 mM malate and 3 different glutamate concentrations.

Also in this work, kinetic relations between substrate consumption and hydrogen utilisation were proposed.

Use of Yeast Extract

Use of yeast extract to replace the standard vitamin solution resulted in an increased rate of hydrogen production (Figure 3) Moreover, the production started earlier.

Figure 3 – Comparison of hydrogen production with yeast extract (0.2 gl) with that of the standard vitamin solution

Use of light-dark cycles

Growth and hydrogen production were minimal in the dark periods but the use of alternating light-dark periods resulted in an increase in hydrogen produced compared to continuous illumination.

Figure 4 – Comparison of hydrogen production in photobioreactor exposed to light-dark cycles (14h-10h) with hydrogen produced in a continuously illuminated photobioreactor

Continuous Hydrogen Production (TOP)

By the addition of bacteria and medium with regular intervals, continuous hydrogen production for long periods, e.g. for more than two months, was accomplished. Figure 5 shows one of these runs. (Eroğlu et. al., 1998)

Figure 5 – Continuous hydrogen production by diluting the reactor with 100 ml of fresh medium and 30 ml bacterial culture every 100 hours. 

Use of Wastewater (TOP)

The cost of the biological hydrogen production can be decreased by supplying at least a part of the nutrient requirements for the bacteria by wastewater. Hydrogen production experiments with the waste water of two common industries-sugar refinery and dairy plant- were conducted.

Use of dairy plant waste water (Türkarslan et. al., 1998) : 

Waste water on its own did not support bacterial growth. A blend of waste water with the standard medium resulted in increased growth and increased hydrogen production compared to the standard medium.

Use of sugar refinery waste water (Yetiş et. al., 2000): 

Hydrogen production was observed in blends of waste water with the standard medium. Highest hydrogen production rate was obtained in 20 % waste water. (Figure 6)

Figure 6 – Hydrogen produced by R. sphaeroides using malate only and a blend of malate and 20 % sugar refinery wastewater.

By-products (TOP)

Identification and isolation of useful products other than hydrogen may increase overall feasibility and provide valuable information on metabolism. Poly-beta-hydroxybutyric acid (PHB), a biodegradable polymer, is one of the by-products of Rhodobacter sphaeroides O.U. 001. PHB can be used for production of disposable bags and containers. Biocompatibility of PHB makes it a desirable material for medical applications such as controlled drug release and the production of surgical pins, wound dressing, blood vessel replacements etc.In R. sphaeroides, PHB is accumulated as an intracellular carbon and energy storage material. PHB is accumulated as granules localized at different sites of cytoplasm. An electron micrograph of PHB granules in R. sphaeroides is seen at the left (Figure 7).

Figure 7. Electron micrograph of PHB granules (*) in Rhodobacter sphaeroides O.U. 001, fixed with Glutaraldehyde and Uranyl acetate, dehydrated with Acetone, stained with Lead citrate.

An example PHB production curve of R. sphaeroides O.U.001 as a function of time is given in Figure 8. R. sphaeroides was found to produce more PHB in a blend of sugar refinery waste water and malate, than from malate alone. This comparison is displayed in Table 2.

Figure 8. Production of PHB as a function of time by Rhodobacter sphaeroides O.U. 001 in malate/ glutamate medium

Table 2: Accumulation of PHB in various media

Metabolism (TOP)

Information on metabolism is essential to understand the mechanisms involved in hydrogen production and to identify the limiting factors. The following information has been obtained from the literature and from conducted experiments. Hydrogen production occurs through the operation of a light-dependent, anaerobic TCA cycle. Electrons produced from the substrate by the TCA cycle are transferred by electron carriers to the nitrogenase. The nitrogenase uses these electrons to reduce protons. 

Flow of electrons: Substrate à(TCA cycle) à NADàFerredoxinàNitrogenase enzyme

Nitrogenase Reaction: 2H⁺ + 2e^- + 4ATPà H₂

R. sphaeroides is capable of several alternative pathways such as aerobic respiration and phototoautotrophy. Hydrogen production is practically absent for these pathways. R. sphaeroides can use a very large variety of carbon and nitrogen sources such as sugars, organic acids, glycerol etc. for growth. However, substrates differ greatly in their manner of utilisation, therefore substrates which promote the hydrogen production pathways are very limited.

Figure 9. Growth curves of R. sphaeroides O.U.001 with: i)Malate as the carbon source(15 mM) and glutamate as the nitrogen source(2 mM), ii) Glutamate (2mM) as the sole carbon and nitrogen source and iii) No carbon source, only ammonium chloride (2mM) supplied.

Figure 9 illustrates the growth of R. sphaeroides under various conditions of carbon availability. Malate+glutamate is the preferred medium. When glutamate is the only carbon and nitrogen source, cells still grow appreciably. When only ammonium chloride is present (no carbon source given) the bacteria still grow to some extent, possibly by utilising their endogeneous reserves, such as PHB.

Conclusions (TOP)

High purity (95-99%) hydrogen has been produced in reactors designed specifically for the purpose. 

Variables such as temperature, light intensity and substrate concentration have been optimized for hydrogen production. 

Kinetic expressions relating substrate consumption and hydrogen production with substrate concentrations have been proposed. 

Experiments have been conducted in which long term continuous hydrogen production has been accomplished. 

Hydrogen production was found to be possible in specific blends of wastewater with the standard medium. 

The valuable by-product poly-hydroxybutyrate has been detected and identified.

Future Plans (TOP)

Improvement of hydrogen production through optimisation of metabolism. For this purpose investigation and identification of the genetic structure is also necessary. 

Construction and continuous operation of an 8 liter outdoor photobioreactor.

Investigation of the effect of other parameters, such as the use of coupled R. sphaeroides-Bacteriorhodopsin systems.

Completion of the characterization and identification of PHB.

Investigation of the possibility of hydrogen production from vegetable oil waste.

References (TOP)

Arık, T., Gündüz, U., Yücel, M., Türker, L., Sediroğlu, V., Eroğlu, İ., "Photoproduction of hydrogen by Rhodobacter sphaeroides O.U.001", Proceedings of the 11th World Hydrogen Energy Conference, Stuttgart, Germany, Vol 3, 2417-2424, 1996 

Biebl, H., Pfennig N., "Isolation of Members of the Family Rhodosprillaceae" M.P. Starr, H. Stolp, H.G. Trüper, A. Balows, H.G. Schlegel (eds.) The Prokaryotes, Springer-Verlag, New York, Vol. 1, 267-273, 1981 

Eroğlu, İ., Aslan, K., Gündüz,U., Yücel, M., Türker, L., "Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor", J. Biotech., 70: 103-113, 1999 

Eroğlu, İ., Aslan, K., Gündüz, U., Yücel, M., Türker, L., "Continuous hydrogen production by Rhodobacter sphaeroides O.U.001.", O.R. Zaborsky (ed.) Biohydrogen, Plenum Press, New York, 143-150, 1998. 

Türkarslan, S., Yiğit, DÖ., Aslan, K., Eroğlu, İ., Gündüz, U., "Photobiological hydrogen production by Rhodobacter sphaeroides O.U.001 by utilization of waste water from milk industry.", O.R. Zaborsky (ed.) Biohydrogen, Plenum Press, New York, 151-156, 1998 

Yetiş, M., Gündüz, U., Eroğlu İ., Yücel, M., Türker, L., "Photoproduction of hydrogen from sugar refinery wastewater by Rhodobacter sphaeroides O.U.001", Int. J. Hyd. Eng., 25: 1035-1041, 2000 

Yiğit, D.Ö., Gündüz, U., Türker, L., Yücel, M., Eroğlu, İ. "Identification of by-products in hydrogen producing bacteria; Rhodobacter sphaeroides O.U.001 grown in the waste water of a sugar refinery." J. Biotech. 70:125-131, 1999.