T I B T E C H - D E C E M B E R 1986

Hydrogen-oxidizing bacteria in biotechnology

Peter Sebo

Using the example of Alcaligenes eutrophus, a lithoautotrophic bacterium, this article surveys the potential fields of application of

the hydrogen-oxidizing bacteria and their products.

substrates can be improved signifi- cantly.

At present there are two advanced projects for the utilization of hydro- gen-oxidizing bacteria biomass: at Imperial Chemical Industries plc in the UK, A. eutrophus is being used to produce 3-hydroxybutyric acid poly- ester and related copolymers ~°,11 and in the Soviet Union the organism is being produced as a feedstock additive 12.

In 1892, Immendorf reported the oxidation of gaseous hydrogen by biological materiaP; 'Knallgas Bak- terien' (bang-gas bacteria) respon- sible for this process were later isolated. The hydrogen-oxidizing bacteria are characterized by their ability to utilize gaseous hydrogen as an electron donor and carbon dioxide as a sole carbon source, i.e. to grow as chemolithoautotrophs 2. Many of them grow in a mixotrophic mode. Until recently, aerobic hydrogen- oxidizing bacteria were considered to be facultative autotrophs. Recently, however, obligatorily autotrophic thermophil ic species have been iso- lated from geothermal areas (e.g. see Ref. 3). The capacity for autotrophic growth is furnished by the presence both of hydrogenases for activating molecular hydrogen and of enzymes of the ribulose bisphosphate cycle for fixation of carbon dioxide. In several species, at least some of these enzymes are coded for on indigenous megaplasmids. An equation for gas consumption and CO2 fixation can be given:

6H2 -F 202 -{- CO2---> (CH20) + 5H20.

Alcaligenes eutrophus, a Gram- negative, motile, rod shaped bac- terium, is the most extensively stud- ied of the heterogeneous group of hydrogen-oxidizing bacteria, and its biotechnological potential has been demonstrated. A. eutrophus has been grown to high cell densities (up to 25 g dry biomass 1-1) in autotrophic batch culture under a controlled atmos- phere of COz, H2 and 02 in a mineral

P. Sebo is at the Institute of Microbiology, Czechoslovak Academy of Sciences, Vide[lsk~ 1083, 142 20 Prague, Czecho- slovakia.

salts medium4; chemostat cultivation has also been successful on a pilot scale 5, and an analytical growth model has been described S. To deliver the potentially explosive mixture of feeding gases more safely, H2 and 02 can be produced by electrolysis of the medium in the culture vessel 7. The rate of gas supply is controlled by regulating the elec- trolytic current. Biomass gasification effluents can also be used as a cheap feedstock for A. eutrophus 8.

Biotechnologies based on hydro- gen-oxidizing bacteria are, in prin- ciple, independent of carbon sources derived from fossil fuels or biomass of photosynthetic origin. In the future, this may favour them over the present technologies based on heterotraphic organisms. Hydrogen is widely considered to be an ecologically acceptable solution to the fuel problem and, therefore, the direct biological transformation of the energy of this fuel to biomass may gain in importance.

Applications of physiological properties of hydrogen-oxidizing bacteria

In batch cultivation of A. eutro- phus, biomass densities up to 25 g of dry mass per liter of culture medium (1011 viable cells m1-1) can be achieved because of the lack of inhibitory by-product formation dur- ing the cultivation 4, Such high yields may ensure the economic profit- abili ty of this method of biomass production. The efficiency of the process can be increased by the use of a closed continuous cultivation sys- tem with recycling of the gaseous feeding mixture 9. By monitoring gas consumption and supplementing the gas mixture accordingly, the efficiency of utilization of gaseous

Polymer production A. eutrophus can accumulate up to

70% of dry biomass as a 3-hydroxy- butyric acid polyester storage poly- mer 1° under conditions of excess carbon and energy with simultan- eous l imitation by another nutrient, preferably the nitrogen source. The thermoplastic polymer has piezo- electric properties applicable in elec- tronics, and its biodegradabili ty may suit it to applications in surgery and to slow release of drugs or agro- chemicals. For these speciality uses, the present cultivation and polymer production processes are already in use commercially. Large tonnage production of the polymer (which is similar in its properties to poly- propylene) is not economical at present13; however, the greater eco- logical acceptability of a biodegrad- able polymer, and possible raw material shortages, make bulk pro- duction of 3-hydroxybutyric acid polyester an attractive goal.

Feedstock additive production The use of hydrogen-oxidizing

bacteria as a feedstock additive has been intensively studied, especially in the Soviet Union. An industrial- scale waste-free process for single cell protein production in a non- sterile fermentation using A. eutro- phus Z-1 has been designed and verified at pilot scale 12. At high growth rates the biomass has a high protein and nucleic acid content. The high nucleic acid content, which can l imit the use of microbial biomass in human diets, may be reduced by heat activation of the endogenous ribo- nuclease: the polymeric RNA con- tents can be decreased from 13% to 1.5% without a protein loss ~4. In cells grown exponentially without nu- trient limitation, up to 75% of the dry

© 1986, Elsevier Science Publishers B.V., Amsterdam 0166- 9430/86/$02.00

T I B T E C H - D E C E M B E R 1986

biomass is protein 15. The protein, more readily digested by the gastro- intestinal enzyme complex than the yeast protein, consists of 40% of essential amino acids and its nu- tritional value is up to 115% of milk casein, the generally used standard. The biomass can also serve as a rich source of vitamins and fatty acids.

When this biomass has been used as a substitute for traditional fodder for livestock (50% of the total), no toxic effects on the animals have been observed and no differences between the animal products (e.g. meat, fats, eggs) and those from conventionally nourished livestock have been de- tected. In contrast to biomass produc- tion from oil-derived substrates, the process is waste-flee and purification of the biomass from metabolic by- products is not necessary (see Ref. 15 for more details). In the Soviet Union, the conversion of methane and gasification of coal could provide the basic raw materials for biomass production on a large scale. By locating fermentation facilities in regions rich in these fuels or near to a power plant, production could be profitable.

Less immediate prospects There are several other possible

applications of hydrogen-oxidizing bacteria: as a source of hydrogenases, for removal of toxic chemicals from the environment, and for production of amino acids.

Production ofhydrogenases Hydrogenases constitute the sole

group of enzymes activating molecu- lar hydrogen without additional energy sources. Their applications have recently been discussed in detail 16.

A. eutrophus produces a soluble cytoplasmic hydrogenase that has several advantages over enzymes from other sources: a very broad spectrum of susceptible substances can be reduced27; it is neither inactivated nor inhibited by oxy- genlS; and for many purposes whole cells can be used as the catalyst. The enzyme is produced not only under autotrophic conditions but also dur- ing growth on fructose, gluconate or citrate (on succinate, lactate or acetate the synthesis is repressed) 19. Hydrogenase synthesis is sometimes

repressed during autotrophic growth in the presence of high concen- trations of oxygen (above 20% in the mixture of gases) 2° but is enhanced under energy-limited conditions.

Several intriguing applications for hydrogenases have been described. Intact cells ofA. eutrophus immobil- ized in calcium alginate can be used as a catalyst for hydrogen-tr i t ium exchange over a broad pH range to detritiate contaminated water from nuclear power plants: 10 g of cells (wet weight) provide catalysis equi- valent to I g of plat inum dioxide 21. In comparison with platinum-based catalysts, the bacterial hydrogenases are potentially inexpensive, readily available in bulk quantities, and completely active in aqueous solu- tion. Their yield from biomass and their activity can be increased severalfold by alteration of culti- vation conditions or genetic manipu- lation 22.

Intact cells and purified A. eutro- phus hydrogenase immobilized to various supports have also been used for regeneration of NADH 23 (used in biocatalytically-performed processes of organic synthesis) and in microbial fuel cells 24. Applicat ion of the reversible high-activity hydrogenase for photochemical production of H2 and fine chemicals in a system with reversed micells is also under in- vestigation 25.

Chemical degradation Some strains of A. eutrophus

can degrade polychlorinated bi- phenyls 26, and cells adsorbed on magnetite have recently been ex- amined for removal of pesticides from water on a laboratory scale 27. The plasmid-coded genes involved in degradation af catechol substances and of some pesticides were trans- ferred to a Rhizobium strain 28. Intro- duction of pesticide-resistant strains of Rhizobia in the field could result in increased fertility of contaminated soil.

Amino acid production A patent on the production of

amino acids from autotrophic cul- tures of hydrogen-oxidizing species of Arthrobacter, Brevibacterium and Mycobacterium has been filed 29 but data on applications are not avail- able.

Genetic aspects ofhydrogen- oxidizingbacteria

The preparation of mutant strains of A. eutrophus and of vectors for gene cloning has been described3°.31; plasmid vector systems based on pRP4, pRK2, pRSF1010 and a cosmid vector based on pSa, mobilizable to A. eutrophus and stably maintained there, have also been described 32-35. However, detailed genetic studies have been restricted to the genes for the key enzymes of autotrophic metabolism, hydrogenase and ribu- lose bisphosphate carboxylase (RuBisCo), and to the pathway for pesticide degradation 36'37.

Genetic manipulat ion of hydrogen- oxidizing bacteria offers two main avenues for exploration by biotech- nologists. The first is the use of these autotrophs as sources of valuable genetic information for improving other organisms; the second is the preparation of mutants for the pro- duction of biomass, biodegradable polymers, amino acids, enzymes and cloned proteins from H2 as a sole energy source and CO2 as a sole carbon source.

S o urces o f gel~ es At Calgene Inc., research on the

RuBisCo genes from A. eutrophus has been in progress for several years: a gene bank has been constructed 32 and the RuBisCo genes transferred to Pseudomonas aeruginosa38; the Ru- BisCo genes have been sequenced and subjected to in vitro mute- genesis 33. The eventual aims of the program include the development of an altered form of RuBisCo lacking oxygenase activity: it is conceivable that substitution of such an enzyme for the RuBisCo in plants could significantly improve crop product- ivity.

Studies on hydrogenase might also play a role in improving agricultural productivity. In several nitrogen- fixing Rhizobium species, a third of the energy consumed in the nitro- genase reaction is lost as gaseous hydrogen 39. Genetic analysis of hydrogen util ization in hydrogen- oxidizing bacteria may open the way for appropriate genetic manipulat ion of important Rhizobium species to remedy this loss 4°. The hydrogenase genes have been localized on mega- plasmid priG1 in A. eutrophus and

T I B T E C H - D E C E M B E R 1986

cloned 41. Genes for chemical degradation

have also been cloned. Those coding for degradation of 2,4-di- chlorophenoxyaceta te were c loned from A. eu t rophus JMP 134 and expressed in E. cel l 42.

Produc t ion organisms Very little work has been per-

formed on improving hydrogen- oxidiz ing bacteria as p roduc t ion organisms but one interest ing pos- sibil i ty is the const ruct ion of protein- secreting strains. The excret ion of c loned proteins has recent ly been demonst ra ted in E. coli 43 and the prospect of a similar system for Gram-negative bacteria wh ich can be grown to high biomass densit ies on s imple minera l media is intriguing.

Conclusion The potent ial of hydrogen-

oxidiz ing bacteria in biotechnologi- cal processes appears to be signifi- cant. However , a lot of work has still to be done. Large-scale cul t ivat ion in cont inuous systems wi th a control led a tmosphere containing high amounts of hydrogen deserves part icular at- tention. Another major topic might be the genetic manipu la t ion and e lucidat ion of the regulatory mech- anisms of autotrophic metabolism.

Future biotechnological processes based on a waste-free, direct conver- s ion of electrical or other forms of energy and inorganic substances to fodder proteins, biological ly act ive substances, biodegradable plastics and other products by means of hydrogen-oxidiz ing bacteria, could make a significant contr ibut ion to the solut ion of global problems facing mankind.

Acknowledgements I thank Dr Richard Bure5 for

an expert computer -a ided literature search, and my colleagues for helpful discussions on the manuscript .

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