**3.4. Enhancement of hydrogen production by lactic acid**

Based on the study of Chojnacka et al. (2011), for the culture containing granular sludge rich in heterolactic bacteria no net production of lactic acid was observed, indicating complete consumption of this metabolite, whereas its concentration in the biofilm-only culture was quite high (Table 4). Noticeable is the fact that molasses - a fermentative substrate in this study, also contains acetic and lactic acids at concentrations of about 800 mg/L each. Furthermore, a significantly higher concentration of butyric acid was detected in the culture containing granular sludge than in biofilm-only culture.

Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 505

in section 4. Therefore, also the biochemical routes leading to butyrate and hydrogen

In the study of Matsumoto and Nishimura (2007) the process of hydrogen production by *C. diolis* from both the slurry solution and a mixture of acetic and lactic acids in an artificial medium occurred to be pH-dependent and was observed in a range of pH (~5.8 – 7.4). Juang et al. (2011) also observed utilization of lactate and acetate for biohydrogen and butyrate production during their studies on hydrogen and methane production from organic residues of ethanol fermentation from tapioca starch by mixed bacteria culture. Lactate and acetate came from maltose fermentation, the main carbohydrate of ethanol fermentation residues. The optimal hydrogen production was observed at pH 5.5 – 6.0. Jo et al. (2008) showed that conversion of lactate and acetate to butyrate and hydrogen by *Clostridium tyrobutyricum* was inhibited due to pH decrease from 5.5 to 4.6. The pH values were dependent on HRT and organic loading rate. At high organic loading rate accumulation of

lactate, pH decrease and a lower efficiency of hydrogen production were observed.

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

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,

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

**intestinal tract** 

2010; Munoz-Tamayo, et al., 2011).

Matsumoto and Nishimura (2007), Jo et al. (2008) and Juang et al. (2011) point to pH values as a critical factor for hydrogen production from lactate and acetate. Various optimal pH for hydrogen production are observed. The differences may depend on the microbial system applied for hydrogen production and the initial substrate. It is speculated that unfavorable changes in pH could be the main reason of inhibiting hydrogen production that could be incorrectly attributed to the presence of lactic acid bacteria in hydrogen-producing consortia. In the study of Chojnacka et al. (2011), the optimal pH was around 5.0. Any change in pH, a decrease below 4.5 or increase above 5.5, caused a significant decline in fermentative gas production. Changes in pH may either be the reason or the results of disturbing the "homeostasis" of hydrogen-producing microbial communities in bioreactors.

production from lactate and acetate are presented in the same section.

There are studies arguing that lactic acid and acetic acid mixed with the substrate stimulate biohydrogen production. Baghchehsaraee et al. (2009) showed that the addition of lactic acid to a mixed culture grown on starch-containing medium increased both hydrogen production and butyric acid formation. Furthermore, complete consumption of lactic acid produced by the culture was observed. When lactic acid was the only carbon source, the level of hydrogen production was very low (0.5% substrate conversion efficiency). Therefore, authors claimed that the addition of lactic acid to the medium probably alters the metabolic pathways in bacterial cells.

In the study of Kim et al. (2012), the effects of different lactate concentrations on hydrogen production from glucose in batch and continuous cultures were examined. Lactic acid was determined to be a factor increasing the efficiency of hydrogen production in a proper range of concentrations. The key issue was to establish the optimal lactic acid concentration. FISH analyses revealed that *Clostridium* sp. was the dominant hydrogen producer in the examined system.

Matsumoto and Nishimura (2007) examined fermentative hydrogen production from sweet potato sho-chu post-distillation slurry that contained large amounts of organic acids. Hydrogen production was accompanied by a decrease in the concentrations of acetic ad lactic acids and co-production of butyric acids. The authors isolated a clostridial strain, *Clostridium diolis* JPCC H-3, capable of effective hydrogen production from the slurry solution and a mixture of acetic and lactic acids in an artificial medium.

The ability to produce hydrogen from lactic and acetic acids seems to be widely conserved in the genus *Clostridium* and other hydrogen-producing bacteria capable of butyric acid fermentation of carbohydrates*.* It was shown that the *Clostridium acetobutylicum* strain P262 and *Butyribacterium methylotrophicum* utilized lactate and acetate and converted them to butyrate, carbon dioxide and hydrogen in the absence of carbohydrates in the medium. Cell extracts from bacteria grown on acetate and lactate showed a higher activity of NADindependent lactate dehydrogenase than these from bacteria grown on carbohydrate-rich medium (Diez-Gonzales et al., 1995; Shen et al., 1996). The authors presented potential biochemical pathways leading to butyrate and hydrogen production from lactate and acetate. Conversion of lactate and acetate to butyrate and symbiotic interactions between LAB and clostridial species in animal intestinal tracts are intensively studied and discussed in section 4. Therefore, also the biochemical routes leading to butyrate and hydrogen production from lactate and acetate are presented in the same section.

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

containing granular sludge than in biofilm-only culture.

metabolic pathways in bacterial cells.

system.

**3.4. Enhancement of hydrogen production by lactic acid** 

Based on the study of Chojnacka et al. (2011), for the culture containing granular sludge rich in heterolactic bacteria no net production of lactic acid was observed, indicating complete consumption of this metabolite, whereas its concentration in the biofilm-only culture was quite high (Table 4). Noticeable is the fact that molasses - a fermentative substrate in this study, also contains acetic and lactic acids at concentrations of about 800 mg/L each. Furthermore, a significantly higher concentration of butyric acid was detected in the culture

There are studies arguing that lactic acid and acetic acid mixed with the substrate stimulate biohydrogen production. Baghchehsaraee et al. (2009) showed that the addition of lactic acid to a mixed culture grown on starch-containing medium increased both hydrogen production and butyric acid formation. Furthermore, complete consumption of lactic acid produced by the culture was observed. When lactic acid was the only carbon source, the level of hydrogen production was very low (0.5% substrate conversion efficiency). Therefore, authors claimed that the addition of lactic acid to the medium probably alters the

In the study of Kim et al. (2012), the effects of different lactate concentrations on hydrogen production from glucose in batch and continuous cultures were examined. Lactic acid was determined to be a factor increasing the efficiency of hydrogen production in a proper range of concentrations. The key issue was to establish the optimal lactic acid concentration. FISH analyses revealed that *Clostridium* sp. was the dominant hydrogen producer in the examined

Matsumoto and Nishimura (2007) examined fermentative hydrogen production from sweet potato sho-chu post-distillation slurry that contained large amounts of organic acids. Hydrogen production was accompanied by a decrease in the concentrations of acetic ad lactic acids and co-production of butyric acids. The authors isolated a clostridial strain, *Clostridium diolis* JPCC H-3, capable of effective hydrogen production from the slurry

The ability to produce hydrogen from lactic and acetic acids seems to be widely conserved in the genus *Clostridium* and other hydrogen-producing bacteria capable of butyric acid fermentation of carbohydrates*.* It was shown that the *Clostridium acetobutylicum* strain P262 and *Butyribacterium methylotrophicum* utilized lactate and acetate and converted them to butyrate, carbon dioxide and hydrogen in the absence of carbohydrates in the medium. Cell extracts from bacteria grown on acetate and lactate showed a higher activity of NADindependent lactate dehydrogenase than these from bacteria grown on carbohydrate-rich medium (Diez-Gonzales et al., 1995; Shen et al., 1996). The authors presented potential biochemical pathways leading to butyrate and hydrogen production from lactate and acetate. Conversion of lactate and acetate to butyrate and symbiotic interactions between LAB and clostridial species in animal intestinal tracts are intensively studied and discussed

solution and a mixture of acetic and lactic acids in an artificial medium.

In the study of Matsumoto and Nishimura (2007) the process of hydrogen production by *C. diolis* from both the slurry solution and a mixture of acetic and lactic acids in an artificial medium occurred to be pH-dependent and was observed in a range of pH (~5.8 – 7.4). Juang et al. (2011) also observed utilization of lactate and acetate for biohydrogen and butyrate production during their studies on hydrogen and methane production from organic residues of ethanol fermentation from tapioca starch by mixed bacteria culture. Lactate and acetate came from maltose fermentation, the main carbohydrate of ethanol fermentation residues. The optimal hydrogen production was observed at pH 5.5 – 6.0. Jo et al. (2008) showed that conversion of lactate and acetate to butyrate and hydrogen by *Clostridium tyrobutyricum* was inhibited due to pH decrease from 5.5 to 4.6. The pH values were dependent on HRT and organic loading rate. At high organic loading rate accumulation of lactate, pH decrease and a lower efficiency of hydrogen production were observed.

Matsumoto and Nishimura (2007), Jo et al. (2008) and Juang et al. (2011) point to pH values as a critical factor for hydrogen production from lactate and acetate. Various optimal pH for hydrogen production are observed. The differences may depend on the microbial system applied for hydrogen production and the initial substrate. It is speculated that unfavorable changes in pH could be the main reason of inhibiting hydrogen production that could be incorrectly attributed to the presence of lactic acid bacteria in hydrogen-producing consortia. In the study of Chojnacka et al. (2011), the optimal pH was around 5.0. Any change in pH, a decrease below 4.5 or increase above 5.5, caused a significant decline in fermentative gas production. Changes in pH may either be the reason or the results of disturbing the "homeostasis" of hydrogen-producing microbial communities in bioreactors.
