**4. Interactions between LAB and clostridial species in the animal intestinal tract**

Microflora of the mammalian intestine is composed of a diverse population of both aerobic and anaerobic bacteria. Symbiotic relationships occur between different intestinal species or groups of species, among which are interactions between LAB and clostridial species. Numerous observations arising from different models describe lactate conversion to butyrate by intestinal bacteria and enhancement of butyrate production by LAB (Hashizume et al., 2003; Duncan et al. 2004; Bourriaud, et al., 2005; Meimandipour et al., 2009; Abbas, 2010; Munoz-Tamayo, et al., 2011).

The microbial community of the human colon contains many bacteria that produce lactic acid including lactobacilli, bifidobacteria, enterococci and streptococci. However, lactate is normally detected only at very low concentration (<5 mM) in feces of healthy individuals due its rapid conversion to short chain fatty acids (SCFAs; acetate, propionate and butyrate) by acid-utilizing bacteria*.* Therefore, lactate is thought to be a precursor of the formation of

various SCFAs (Hashizume et al., 2003; Duncan et al., 2004; Bourriaud et al., 2005; Munoz-Tamayo et al., 2011). Bourriaud and colleagues (2005) performed convincing experiments exploring the lactate metabolism and short fatty acids production. They incubated three human microfloras with media containing 13C-labelled lactate and detected the labeled products of fermentation by 13C NMR spectrometry. Results revealed that butyrate was the major net product of lactate conversion by human fecal microflora. Other SCFAs produced were: propionate, acetate and valerate. Inter-individual differences between the three microfloras were observed. Similar studies performed using 2H-labelled acetate and 13Clabelled lactate and gas chromatography-mass spectrometry (GC-MS) analysis showed that acetic and lactic acids are important precursors of butyrate production in human fecal samples (Morrison et al., 2006).

Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 507

mammals. However, only a few butyrate-producing species within the clostridial cluster XIVa are capable of converting lactate to butyrate (Duncan, 2004; Hashizume et al., 2003;

**Figure 6.** Scheme for butyrate production from lactate in *E. hallii* and *A. caccae*, adapted from Duncan et

The issue of stereospecificity of lactate utilization was addressed in the study of Duncan et al. (2004). Three *E. hallii*-related strains (SL6/1/1, SM6/1 and L2-7) and two *A. caccae* strains (L1-92 and P2) were able to use both D and L isomers of lactate during incubation on DLlactate-containing medium. Interestingly, the addition of glucose to the medium almost

Munoz-Tamayo, et al., 2011).

al., 2004.

The metabolic pathway of lactate and acetate utilization to produce butyrate proposed for *Eubacterium hallii* and *Anaerostipes caccae* is shown on Figure 6 (Duncan, et al., 2004; Munoz-Tamayo, et al., 2011). The butyrate produced (in moles) is approximately equal to the sum of half of the acetate and lactate coming from the medium. Lactate is converted to pyruvate by lactate dehydrogenase. The next steps are analogous to those ones presented on Figure 1. Pyruvate is oxidized to acetyl coenzyme A (acetyl-CoA), which is further routed to acetate and butyrate. Acetate is produced via acetate kinase, the pathway generating energy in the form of ATP. For butyrate formation, two molecules of acetyl-CoA are condensed to one molecule of acetoacetyl-CoA, and subsequently reduced to butyryl-CoA. Butyrate can be synthesized from two metabolic pathways: phosphotransbutyrylase and butyrate kinase as shown on Figure 1, and butyryl CoA:acetate CoA transferase as shown on Figure 6. The latter mechanism seems to be the dominant in the human colonic ecosystem. Butyryl-CoA:acetate CoA-transferase transports the CoA component to exterior of acetate releasing butyrate and acetyl-CoA (Duncan et al., 2004; Munoz-Tamayo, et al., 2011). Hydrogen can be produced by both PFOR and NFOR complexes and hydrogenases, as described in section 2. The reaction catalyzed by NFOR is assumed to be the main route for H2 production by intestinal microflora (Bourriaud et al., 2005). Similar pathway is proposed for clostridial species (eg. *C. acetobutylicum*; Diez-Gonzales et al., 1995) and other hydrogen and butyrate producing bacteria (eg. *B. methylotrophicum*; Shen et al., 1996), as mentioned in section 3.4. Conversion of lactate and acetate to butyrate and hydrogen is an energetically favorable process (Duncan et al., 2004; Jo et al., 2008).

The known lactate-utilizing butyrate-producing bacteria belong to the *Firmicutes* phylum, which includes the following species: *Megasphaera elsdenii, Anaerostipes caccae*, *Anaerostipes coli*, *E. hallii* and species distantly related to *Clostridium indolis*. *A. coli* is a dominant member of the human colonic microbiota recognized for its importance in butyrate production. *M. elsdenii* is one of the main butyrate producers from lactate in ruminants as well as monogastric animals, such as pigs or rodents. *A. caccae*, *A. coli*, *E. hallii* and species distantly related to *Clostridium indolis* belong to the clostridial cluster XIVa (*Lachnospiraceae*), known butyrate-producing bacteria of gastrointestinal tracts in mammals. However, only a few butyrate-producing species within the clostridial cluster XIVa are capable of converting lactate to butyrate (Duncan, 2004; Hashizume et al., 2003; Munoz-Tamayo, et al., 2011).

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

samples (Morrison et al., 2006).

process (Duncan et al., 2004; Jo et al., 2008).

various SCFAs (Hashizume et al., 2003; Duncan et al., 2004; Bourriaud et al., 2005; Munoz-Tamayo et al., 2011). Bourriaud and colleagues (2005) performed convincing experiments exploring the lactate metabolism and short fatty acids production. They incubated three human microfloras with media containing 13C-labelled lactate and detected the labeled products of fermentation by 13C NMR spectrometry. Results revealed that butyrate was the major net product of lactate conversion by human fecal microflora. Other SCFAs produced were: propionate, acetate and valerate. Inter-individual differences between the three microfloras were observed. Similar studies performed using 2H-labelled acetate and 13Clabelled lactate and gas chromatography-mass spectrometry (GC-MS) analysis showed that acetic and lactic acids are important precursors of butyrate production in human fecal

The metabolic pathway of lactate and acetate utilization to produce butyrate proposed for *Eubacterium hallii* and *Anaerostipes caccae* is shown on Figure 6 (Duncan, et al., 2004; Munoz-Tamayo, et al., 2011). The butyrate produced (in moles) is approximately equal to the sum of half of the acetate and lactate coming from the medium. Lactate is converted to pyruvate by lactate dehydrogenase. The next steps are analogous to those ones presented on Figure 1. Pyruvate is oxidized to acetyl coenzyme A (acetyl-CoA), which is further routed to acetate and butyrate. Acetate is produced via acetate kinase, the pathway generating energy in the form of ATP. For butyrate formation, two molecules of acetyl-CoA are condensed to one molecule of acetoacetyl-CoA, and subsequently reduced to butyryl-CoA. Butyrate can be synthesized from two metabolic pathways: phosphotransbutyrylase and butyrate kinase as shown on Figure 1, and butyryl CoA:acetate CoA transferase as shown on Figure 6. The latter mechanism seems to be the dominant in the human colonic ecosystem. Butyryl-CoA:acetate CoA-transferase transports the CoA component to exterior of acetate releasing butyrate and acetyl-CoA (Duncan et al., 2004; Munoz-Tamayo, et al., 2011). Hydrogen can be produced by both PFOR and NFOR complexes and hydrogenases, as described in section 2. The reaction catalyzed by NFOR is assumed to be the main route for H2 production by intestinal microflora (Bourriaud et al., 2005). Similar pathway is proposed for clostridial species (eg. *C. acetobutylicum*; Diez-Gonzales et al., 1995) and other hydrogen and butyrate producing bacteria (eg. *B. methylotrophicum*; Shen et al., 1996), as mentioned in section 3.4. Conversion of lactate and acetate to butyrate and hydrogen is an energetically favorable

The known lactate-utilizing butyrate-producing bacteria belong to the *Firmicutes* phylum, which includes the following species: *Megasphaera elsdenii, Anaerostipes caccae*, *Anaerostipes coli*, *E. hallii* and species distantly related to *Clostridium indolis*. *A. coli* is a dominant member of the human colonic microbiota recognized for its importance in butyrate production. *M. elsdenii* is one of the main butyrate producers from lactate in ruminants as well as monogastric animals, such as pigs or rodents. *A. caccae*, *A. coli*, *E. hallii* and species distantly related to *Clostridium indolis* belong to the clostridial cluster XIVa (*Lachnospiraceae*), known butyrate-producing bacteria of gastrointestinal tracts in

**Figure 6.** Scheme for butyrate production from lactate in *E. hallii* and *A. caccae*, adapted from Duncan et al., 2004.

The issue of stereospecificity of lactate utilization was addressed in the study of Duncan et al. (2004). Three *E. hallii*-related strains (SL6/1/1, SM6/1 and L2-7) and two *A. caccae* strains (L1-92 and P2) were able to use both D and L isomers of lactate during incubation on DLlactate-containing medium. Interestingly, the addition of glucose to the medium almost

completely inhibited lactate utilization by the tested strains. Additional studies showed that *E. hallii* L2-7, when grown with DL-lactate, used all of the supplied lactic acid together with some acetate, producing more than 20 mM of butyrate. Less butyrate, but a noteworthy amount of formate, was produced during growth on glucose or on glucose plus lactate. Interestingly, the highest level of hydrogen production was observed when strains were grown on lactate and the lowest for growth on glucose plus lactate. However, the *Clostridium indolis*-related strain SS2/1 was able to use D-lactate, but not L-lactate, during growth on DL-lactate containing media, which suggests that it lacks both an L-lactate dehydrogenase, capable of producing pyruvate from L-lactate, and a racemase, capable of converting L-lactate into D-lactate. According to Bourriaud and colleagues (Bourriaud et al., 2005), both lactate enantiomers are equally utilized by human intestinal microflora, treated as a whole consortium, not as pure strains.

Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 509

production due to substrate competition and replacement of hydrogen fermentation by lactic acid and ethanol fermentations, and/or excretion of bacteriocines. On the other hand, some positive interactions between LAB and clostridial species have also been noted. They include hydrogen production from lactate by many clostridial species and symbiotic

These phenomena rely on the conversion of lactate and acetate to butyrate and hydrogen. Symbiotic interactions between LAB and butyrate-producing bacteria involving clostridia have been described in the gastrointestinal tract. We postulate that similar relations exist in biohydrogen-producing bioreactors. According to our hypothesis, pH may be a critical factor affecting bacterial growth, development of specific groups building hydrogenproducing microbial communities and fermentation processes. Acidity changes in bioreactors might be either the reason or the results of disturbances in the balance between microorganisms constituting hydrogen-producing microbial communities in bioreactors. Still, there are no data on symbiotic interactions between LAB and enterobacteria in hydrogen-producing microbial consortia. All these issues require further investigations.

interactions, called lactate cross-feeding, occurring between LAB and clostridia.

*Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland* 

*Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Poland* 

We would like to thank Dominika Brodowska for performance of FISH analyses and Dr.

Abbas, K.A. (2010). The synergistic effects of probiotic microorganisms on the microbial production of butyrate in vitro, *McNair Scholars Research Journal* 2(1): 103-113. Angenent, L.T., Karim, K., Al-Dahhan, M.H., Wrenn, B.A. & Domiguez-Espinoza, R. (2004). Production of bioenergy and biohemicals from industrial and agricultural wastewater.

Antoni, D., Zverlov, V.V. & Schwarz, W.H. (2007). Biofuels from microbes. *Appl. Microbiol.* 

Baghchehsaraee, B., Nakla, G., Karamanev, D., Margaritis, A. & Reid, G. (2008). The effect of heat pretreatment temperature on fermentative hydrogen production using mixed

**Author details** 

**Acknowledgement** 

**6. References** 

Anna Sikora and Urszula Zielenkiewicz

Mieczysław Błaszczyk and Marcin Jurkowski

Agnieszka Szczepankowska for editorial assistance.

*TRENDS Biotechnol.* 22 (9): 477-485.

cultures. *Int. J. Hydrogen Energy* 33(15): 4064-4073.

*Biotechnol.* 77(1): 23–35.

Conversion of lactate to butyrate is one of the important factors for maintaining homeostasis in gastrointestinal tracts. Accumulation of lactate leads to different intestinal disorders (Hashizume et al., 2003). A number of studies have been performed to confirm the symbiotic interaction between lactic acid bacteria and butyric acid bacteria, mainly the *Clostridiales*  representatives isolated from animal gastrointestinal tracts. Co-culture experiments that simulated the relations occurring *in vivo* were carried out. Symbiotic interactions were described to rely on the phenomenon of cross-feeding of lactate and involve conversion of lactate to butyrate by butyrate-producing bacteria stimulated by LAB.

It is noteworthy that results from studies of the gastrointestinal microflora indicate that acidity seems to be a key regulatory factor in lactate metabolism. The pH values may influence both bacterial growth and development of specific groups of bacteria as well as fermentation processes affecting the relative proportions of SCFAs (Belenguer et al., 2006; Meimandipour, et al., 2009; Belenguer et al., 2011). These observations are in agreement with our position concerning the potentially important role of pH in hydrogen-producing consortia discussed in section 3.4.

We postulate that the phenomenon analogous to cross-feeding observed in the gastrointestinal tract might take place in hydrogen-producing bioreactors. Although LAB may seem to be undesirable in such processes as they use some of H2 to produce lactate, their stimulatory effects on hydrogen producers seem to exceed the potentially unbeneficial features. In many studies, it has been explicitly proven that the presence of LAB positively affects the production of butyrate. Most probably, hydrogen producers, mainly species belonging to the *Clostridiales* order, are capable of utilizing lactate as the main precursor of butyrate formation. Further investigations are required.

## **5. Conclusions**

Lactic acid bacteria are detected in almost all biohydrogen-producing microbial communities of dark fermentation. Many studies indicate that LAB inhibit hydrogen production due to substrate competition and replacement of hydrogen fermentation by lactic acid and ethanol fermentations, and/or excretion of bacteriocines. On the other hand, some positive interactions between LAB and clostridial species have also been noted. They include hydrogen production from lactate by many clostridial species and symbiotic interactions, called lactate cross-feeding, occurring between LAB and clostridia.

These phenomena rely on the conversion of lactate and acetate to butyrate and hydrogen. Symbiotic interactions between LAB and butyrate-producing bacteria involving clostridia have been described in the gastrointestinal tract. We postulate that similar relations exist in biohydrogen-producing bioreactors. According to our hypothesis, pH may be a critical factor affecting bacterial growth, development of specific groups building hydrogenproducing microbial communities and fermentation processes. Acidity changes in bioreactors might be either the reason or the results of disturbances in the balance between microorganisms constituting hydrogen-producing microbial communities in bioreactors. Still, there are no data on symbiotic interactions between LAB and enterobacteria in hydrogen-producing microbial consortia. All these issues require further investigations.
