**1. Introduction**

486 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

*of Bacteriology*, Vol. 189, No. 4, pp. 1366-81, ISSN 0021-9193

1755-68, ISSN 0950-382X

galactosidase in *Agrobacterium radiobacter*. *Molecular Microbiology*, Vol. 6, No. 13, pp.

Zomer, AL., Buist, G., Larsen, R., Kok, J. & Kuipers, OP. (2007). Time-resolved determination of the CcpA regulon of *Lactococcus lactis* subsp. *cremoris* MG1363. *Journal* 

> Hydrogen is both a valuable energy carrier and a feedstock for various branches of the chemical industry. It is thought to be one of the most important energy carriers of the future, an alternative to conventional fossil fuels. Water vapor and heat energy are the sole products of hydrogen burning. Therefore, the use of hydrogen to generate energy does not contribute to ozone depletion, the greenhouse effect, climate changes or acid rains. Hydrogen is a highly efficient energy source; its specific energy equals 33 Wh/g, which is the highest among all fuels. For comparison, the specific energy of methane is 14.2 Wh/g and coal, 9.1 Wh/g. Hydrogen can be used as a fuel in hydrogen fuel cells or burn directly in internal combustion engines. In the chemical industry, hydrogen is used for syntheses of ammonia, alcohols, aldehydes, hydrogen chloride and for the hydrogenation of edible oils, heavy oils or ammonia, for removal of oxygen traces in prevention against metal oxidation and corrosion processes (Nath & Das, 2003; Logan, 2004; Antoni et al., 2007; Piela & Zelenay, 2004).

> Conventional methods of hydrogen production, such as gasification of coal, steam reforming of natural gas and petroleum, and electrolysis of water, are based on fossil fuels. Therefore, these methods are regarded as energy expensive and cause environmental pollution (Nath & Das, 2003; Logan, 2004; Nath & Das, 2004).

> Considering the limited reserves of fossil fuels, environmental pollution and global warming, there is great interest in biological methods of producing fuels, such as bio-hydrogen, biogas (methane), ethanol or diesel. Among the known biological processes leading to hydrogen production are dark fermentation, photofermentation, direct and indirect biophotolysis, as well as anaerobic respiration of sulphate-reducing bacteria under conditions of sulphate depletion. Taking under account potential applications, microbial hydrogen production has

© 2013 Zielenkiewicz et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

been focused on: (i) photolysis of water using algae and *Cyanobacteria*, (ii) photofermentation of organic compounds by photosynthetic bacteria, and (iii) dark fermentation of organic compounds using anaerobic bacteria.

Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 489

Pyruvate CoA Fd acetyl CoA FdH CO (1)

2

(2)

Reduced ferredoxin is also formed in the reaction with NADH catalyzed by

 NFOR NADH Fd NAD FdH

Hydrogen is released by hydrogenases that catalyze proton reduction using electrons from ferredoxin. The activity of PFOR and NFOR enzymes is thermodynamically regulated by the hydrogen concentration. Partial hydrogen pressure >60 Pa inhibits the NFOR activity and favors formation of non-gaseous end-products from acetyl-CoA including acetate, butyrate, ethanol, butanol and lactate. PFOR is active at hydrogen concentrations up to 3×104 Pa (Angenent et al., 2004; Girbal et al., 1995; Hallenbeck, 2005; Kraemer & Bagley, 2007; Lee et

The theoretical maximum hydrogen yield during clostridial-type fermentation is 4 moles of hydrogen per mole of glucose, when all of the substrate is converted to acetic acid (See

This gives the maximal possible level of hydrogen yield during dark fermentation. When the glucose is converted to butyrate the hydrogen yield drops to 2 moles (See Equation 4).

Formation of other non-gaseous end products of fermentation causes further decrease in hydrogen yields. The scheme of the clostridial-type fermentation is presented in Figure 1

The described type of fermentation is the most characteristic for spore-forming representatives of the *Clostridium* as well as *Bacillus* genera and others, such as the rumen bacteria e.g. *Ruminococcus albus*. Among the fermentative anaerobes, clostridia have been well known and extensively studied for their capability to produce hydrogen from various carbohydrates (Kalia & Purohit, 2008; Lee et al., 2011). The hydrogen yields of pure *Clostridium* cultures, including *C. acetobutylicum*, *C. bifermentans*, *C. butyricum*, *C. kluyveri*, *C. lentocellum*, *C. paraputrificum*, *C. pasteurianum*, *C. saccharoperbutylacetonicum*, *C. thermosuccinogenes*, and *C. thermolacticum* were examined. The optimum hydrogen yields observed for these bacteria varied between 1.1 moles of H2/mole of hexose and 2.6 moles of H2/mole of -hexose, dependent on the organism per se as well as environmental conditions

C H O 2 H O 4 H 2 CO 2 CH COOH 6 12 6 2 <sup>2</sup> <sup>2</sup> <sup>3</sup> (3)

C H O 2 H O 2 H 2 CO CH CH CH COOH 6 12 6 2 <sup>2</sup> 2 322 (4)

PFOR

NADH:ferredoxin oxidoreductase (NFOR) (See Equation 2).

al., 2011).

Equation 3).

(Papoutsakis, 1984; Saint-Amans et al., 2001).

(for review see Lee et al., 2011).

Members of the *Clostridiales* and *Enterobacteriaceae* are well-recognized hydrogen-producers during the process of dark fermentation. For future applications, dark fermentation seems to be the most promising concept. However, low hydrogen yields and generation of large quantities of non-gaseous organic products remain key problems of dark fermentation. The theoretical maximum hydrogen yield during dark fermentation is 4 moles of H2/mole of glucose (~33% substrate conversion), but the actual yield is only 2 moles of H2/mole of glucose (~17% conversion). Currently, many investigations are focused on improving the hydrogen yield during fermentation as an alternative method of hydrogen production and combining dark fermentation with other processes, like methanogenesis, photofermentation or microbial electrolysis of cells, to achieve more effective substrate utilization (Li & Fang, 2007; Das & Veziroglu, 2008; Hallenbeck & Ghosh, 2009; Lee et al., 2010; Hallenbeck, 2011). Biohydrogen fermentations may be carried out in different batch types, continuous or semi-continuous bioreactors, where mixed microbial consortia develop. In the most effective systems, consortia are selected for growth and dominance under non-sterile conditions and usually show high stability and resistance to transient unfavorable changes in the bioreactor environment. Depending on the bioreactor type and growth conditions, consortia form various structures which ensure retention and accumulation of the active biomass. These include microbial-based biofilms and macroscopic aggregates of microbial cells, such as flocs and granules (Campos et al., 2009; Hallenbeck & Ghosh, 2009). A good understanding of the structure of hydrogenproducing microbial communities, symbiotic relationships within the consortia as well as factors favoring hydrogen production is vital for optimizing the process.

Interestingly, lactic acid bacteria (LAB) are often detected in mesophilic hydrogenproducing consortia as bacteria that accompany hydrogen producers. In this chapter, we discuss the issue of whether LAB are bad or good (positive or negative) components of hydrogen-producing consortia. We present different opinions about the potential significance and the role of LAB in hydrogen-producing communities.
