**2. Hydrogen-producing bacteria**

Fermentation is an anaerobic type of metabolic process of low energy gain in which organic compounds are degraded in the absence of external electron acceptors and a mixture of oxidized and reduced products are formed. Products, namely organic compounds and gasses (hydrogen and carbon dioxide), determine the type of fermentation. Main hydrogen yielding fermentations are butyric acid fermentation (saccharolytic clostridial-type fermentation) and mixed-acid fermentation (enterobacterial-type fermentation). The first step of both fermentations is the Embden-Meyerhof pathway or glycolysis in which glucose is converted into pyruvate and NADH is formed.

In the clostridial-type fermentation pyruvate is oxidized to acetyl-CoA by pyruvate:ferredoxin oxidoreductase (PFOR) in the presence of ferredoxin (Fd) (See Equation 1).

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

$$\begin{aligned} \text{(PFOR)}\\ \text{Pyruvate + CoA + Fd \rightarrow acetyl-CoA + FdH + CO}\_2 \end{aligned} \tag{1}$$

Reduced ferredoxin is also formed in the reaction with NADH catalyzed by NADH:ferredoxin oxidoreductase (NFOR) (See Equation 2).

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

compounds using anaerobic bacteria.

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

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.

significance and the role of LAB in hydrogen-producing communities.

oxidoreductase (PFOR) in the presence of ferredoxin (Fd) (See Equation 1).

**2. Hydrogen-producing bacteria** 

is converted into pyruvate and NADH is formed.

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

Fermentation is an anaerobic type of metabolic process of low energy gain in which organic compounds are degraded in the absence of external electron acceptors and a mixture of oxidized and reduced products are formed. Products, namely organic compounds and gasses (hydrogen and carbon dioxide), determine the type of fermentation. Main hydrogen yielding fermentations are butyric acid fermentation (saccharolytic clostridial-type fermentation) and mixed-acid fermentation (enterobacterial-type fermentation). The first step of both fermentations is the Embden-Meyerhof pathway or glycolysis in which glucose

In the clostridial-type fermentation pyruvate is oxidized to acetyl-CoA by pyruvate:ferredoxin

$$\begin{aligned} \text{(NFOR)}\\ \text{NADH} + \text{Fd} \rightarrow \text{NAD}^+ + \text{FdH} \end{aligned} \tag{2}$$

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 al., 2011).

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 Equation 3).

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 + 2\text{ H}\_2\text{O} \rightarrow 4\text{ H}\_2 + 2\text{ CO}\_2 + 2\text{ CH}\_3\text{COOH} \tag{3}$$

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).

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 + 2\text{ H}\_2\text{O} \rightarrow 2\text{ H}\_2 + 2\text{ CO}\_2 + \text{CH}\_3\text{CH}\_2\text{CH}\_2\text{COOH} \tag{4}$$

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

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

Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 491

Formic acid HCOOH H CO (6)

Pyruvate CoA acetyl CoA formic acid (5)

 2 2 FHL

The formic acid can be degraded into hydrogen and carbon dioxide by formate hydrogen-

There are two types of mixed-acid fermentations. In the first type ethanol and a complex mixture of acids, particularly acetic, lactic, succinic and formic acids are produced. This pattern is seen in *Escherichia*, *Salmonella*, *Proteus* and other genera. The second type is characteristic for *Enterobacter*, *Serratia*, *Erwinia* and some species of *Bacillus*. In this type of

The theoretical hydrogen yields during mixed acid fermentation are lower than those described for the clostridial-type fermentation. Hydrogen yields of *Escherichia* spp., as obtained for the pure culture of *E. coli* NCIMB 11943, are in a range of 0.2–1.8 moles of H2/mole of hexose, when glucose or starch hydrolysate are substrates, whereas hydrogen yields determined for pure *Enterobacter* spp. cultures are much higher, ranging from 1.1 moles of H2/mole of hexose to ca. 3.0 moles of H2/mole of hexose (Lee et al., 2011). It is known that in the *Enterobacter*-type fermentation hydrogen is also generated through oxidation of NADH by NFOR in reactions similar to those described for the clostridial-type

Lactic acid bacteria are Gram-positive bacteria, producing lactic acid as the main product of carbohydrate fermentation. Two types of lactic acid fermentation are distinguished: homolactic and heterolactic fermentation. In homolactic acid fermentation, two molecules of pyruvate that are formed during glycolysis are converted to lactate. In heterolactic acid fermentation, one molecule of pyruvate is converted to lactate; the other is converted to

At present, nearly 400 LAB species have been recognized. They include bacteria belonging to the order *Lactobacillales* classified into seven families: *Lactobacillaceae* (genera: *Lactobacillus* and *Pediococcus*); *Aerococcaceae* (genus *Aerococcus*); *Carnobacteriaceae* (genera: *Alloiococcus*, *Carnobacterium*, *Dolosigranulum***,** *Granulicatella* and *Lactosphaera*); *Enterococcaceae* (genera: *Enterococcus*, *Tetragenococcus* and *Vagococcus*); *Leuconostocaceae* (genera: *Leuconostoc*, *Oenococcus* and *Weisella*); *Streptococcaceae* (genera: *Streptococcus*, *Lactococcus* and *Melissococcus*); *Microbacteriaceae* (genus *Microbacterium*). Extremely varied among lactic acid

fermentation, acetoin, 2,3-butanediol, ethanol and lower amount of acids are formed.

fermentation (Nakashimada et al., 2002; Sawers, 2005; Maeda et al., 2007).

The pathway of the mixed-acid fermentation is presented in Figure 2.

**3. Lactic acid bacteria in hydrogen-producing consortia** 

**3.1. Lactic acid bacteria – General information** 

ethanol and carbon dioxide.

PFL

lyase (FHL) (See Equation 6).

**Figure 1.** The scheme of clostridial-type fermentation. The pathway leading to the theoretical maximum hydrogen yield of 4 moles of hydrogen per 1 mole of glucose, when all of the substrate is converted to acetic acid is labeled in red.

In the mixed acid-fermentation (also known as formic acid fermentation) pyruvate formatelyase (PFL) converts pyruvate to acetyl-CoA and formic acid (See Equation 5).

$$\begin{array}{c} \text{PFL} \\ \text{Pyruvate + CoA} \rightarrow \text{acetyl-CoA + formic acid} \end{array} \tag{5}$$

The formic acid can be degraded into hydrogen and carbon dioxide by formate hydrogenlyase (FHL) (See Equation 6).

$$\begin{aligned} \text{FHL} \\ \text{Formic acid (HCOOH)} \rightarrow \text{H}\_2 + \text{CO}\_2 \end{aligned} \tag{6}$$

There are two types of mixed-acid fermentations. In the first type ethanol and a complex mixture of acids, particularly acetic, lactic, succinic and formic acids are produced. This pattern is seen in *Escherichia*, *Salmonella*, *Proteus* and other genera. The second type is characteristic for *Enterobacter*, *Serratia*, *Erwinia* and some species of *Bacillus*. In this type of fermentation, acetoin, 2,3-butanediol, ethanol and lower amount of acids are formed.

The theoretical hydrogen yields during mixed acid fermentation are lower than those described for the clostridial-type fermentation. Hydrogen yields of *Escherichia* spp., as obtained for the pure culture of *E. coli* NCIMB 11943, are in a range of 0.2–1.8 moles of H2/mole of hexose, when glucose or starch hydrolysate are substrates, whereas hydrogen yields determined for pure *Enterobacter* spp. cultures are much higher, ranging from 1.1 moles of H2/mole of hexose to ca. 3.0 moles of H2/mole of hexose (Lee et al., 2011). It is known that in the *Enterobacter*-type fermentation hydrogen is also generated through oxidation of NADH by NFOR in reactions similar to those described for the clostridial-type fermentation (Nakashimada et al., 2002; Sawers, 2005; Maeda et al., 2007).

The pathway of the mixed-acid fermentation is presented in Figure 2.

### **3. Lactic acid bacteria in hydrogen-producing consortia**

### **3.1. Lactic acid bacteria – General information**

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

**Figure 1.** The scheme of clostridial-type fermentation. The pathway leading to the theoretical maximum hydrogen yield of 4 moles of hydrogen per 1 mole of glucose, when all of the substrate is converted to

In the mixed acid-fermentation (also known as formic acid fermentation) pyruvate formate-

lyase (PFL) converts pyruvate to acetyl-CoA and formic acid (See Equation 5).

acetic acid is labeled in red.

Lactic acid bacteria are Gram-positive bacteria, producing lactic acid as the main product of carbohydrate fermentation. Two types of lactic acid fermentation are distinguished: homolactic and heterolactic fermentation. In homolactic acid fermentation, two molecules of pyruvate that are formed during glycolysis are converted to lactate. In heterolactic acid fermentation, one molecule of pyruvate is converted to lactate; the other is converted to ethanol and carbon dioxide.

At present, nearly 400 LAB species have been recognized. They include bacteria belonging to the order *Lactobacillales* classified into seven families: *Lactobacillaceae* (genera: *Lactobacillus* and *Pediococcus*); *Aerococcaceae* (genus *Aerococcus*); *Carnobacteriaceae* (genera: *Alloiococcus*, *Carnobacterium*, *Dolosigranulum***,** *Granulicatella* and *Lactosphaera*); *Enterococcaceae* (genera: *Enterococcus*, *Tetragenococcus* and *Vagococcus*); *Leuconostocaceae* (genera: *Leuconostoc*, *Oenococcus* and *Weisella*); *Streptococcaceae* (genera: *Streptococcus*, *Lactococcus* and *Melissococcus*); *Microbacteriaceae* (genus *Microbacterium*). Extremely varied among lactic acid

bacteria is genus *Lactobacillus* which comprises over 145 species. Genera *Bifidobacterium* and *Propionibacterium* (class: *Actinobacteria*) as well as spore forming rods belonging to the order *Bacillales*, family *Sporolactobacillaceae*, genus *Sporolactobacillus* constitute further groups of LAB. With the exception of bacteria belonging to the genera *Lactobacillus*, *Carnabacterium*, *Weissella* and *Sporolactobacillus* which are rods, other species of lactic acid bacteria are cocci (de Vos et al., 2009).

Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 493

of lactic acid bacteria colonizing the human digestive system is high; however, the species composition is constantly changing as most of the species colonize the gastrointestinal tract for only a short period (Korhonen, 2010). Microorganisms in the adult intestine outnumber by 10-fold cells constituting the human body. The microbial composition for each individual is unique, depending on age, diet, diseases and environmental factors (Qin J. et al., 2010). LAB have been widely used as probiotic bacteria in the human gastrointestinal tract,

The natural occurrence of lactic acid bacteria on plants (fruits, vegetables and grains) as well as in milk permitted their use in biotechnology (Makarova et al., 2006). *Lactobacillus, Pediococcus, Leuconostoc* and *Oenococcus* which reside on grapes, enable fruit fermentation and wine production (de Nadra, 2007). Also, LAB can occur naturally or be intentionally added as starter cultures during plant, meat and dairy fermentation (Korhonen, 2010).

In marine environments LAB play a role in the breakdown of organic matter. In the last decade LAB belonging to the following genera: *Amphibacillus*, *Alkalibacterium*, *Marinilactibacillus*, *Paraliobacillus*, *Halolactibacillus* were isolated from the samples taken from the sea and oceanic as well as from animals that inhabit these ecosystems. These bacteria

Interestingly, lactic acid bacteria are often detected in mesophilic hydrogen-producing consortia as bacteria that accompany hydrogen producers. The technique most commonly used for analyzing the diversity of hydrogen-producing microbial communities is polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), followed by either direct sequencing or cloning and sequencing of DGGE bands. One of the disadvantages of this method is underestimation of the true bacterial diversity due to the fact that only the most prominent DGGE bands are analyzed. Various studies have shown that DGGE bands representing LAB are one of the most dominant bands (Fang et al., 2002; Kim et al., 2006; Li et al., 2006; Wu et al., 2006; Hung et al., 2007; Ren et al., 2007; Jo et al., 2007; Lo et al., 2008; Sreela-or et al., 2011). Another method of analyzing the biodiversity of hydrogen-producing consortia is cloning and sequencing of the 16S rDNA gene amplified on the total DNA isolated from the culture probes. Also with this method, sequences related to lactic acid bacteria have been detected (Yang et al., 2007). An alternative method used by our group for the first time to perform metagenomic analysis of hydrogen-producing microbial communities is 454-pyrosequencing. Our results showed that *Clostridiaceae, Enterobacteriaceae* and heterolactic fermentation bacteria, mainly *Leuconostocaeae,* were the most dominant bacteria in hydrogen-producing consortia under optimal condition for gas

The aim of the chapter is a provocative discussion on the true role of LAB in hydrogenproducing bioreactors and their influence on hydrogen producers. Table 1 presents a set of selected studies which examine the possible influence of lactic acid bacteria on hydrogen

contributing to pathogen inhibition and immunomodulation (Zhang et al., 2011).

were named "marine LAB" (Ishikawa et al., 2005).

production (Chojnacka et al., 2011).

production during dark fermentation.

**3.2. Lactic acid bacteria – Influence on hydrogen producers** 

LdhA – lactate dehydrogenase, PoxB – pyruvate oxydase, PTA – phosphotransacetylase, ACK – acetate kinase, PFL – pyruvate formate lyase, FHL – formate hydrogen lyase.

**Figure 2.** The scheme of mixed-acid fermentation (*Escherichia coli*-type). The pathway leading to hydrogen production is shown in red.

LAB are microorganisms ubiquitous in the environment. Due to their high nutritional requirements, they are usually found in environments rich in carbohydrates, amino acids and nucleotides. On the other hand, they show considerable adaptation to the harsh conditions, which allows them to inhabit a range of various niches (Korhonen, 2010).

The digestive tracts of man and animals are among the environments where LAB occur. They have been reported in saliva, the small intestine and colon (Korhonen, 2010). The development of the gastrointestinal microflora in infants is influenced by contact with diverse microflora of the mother and of the closest surrounding. The main species found in both infants and adults are *Lactobacillus ruminis, L. salivarius, L. gasseri*, *L. reuteri* as well as *Bifidobacterium longum* and *B. breve* (Salminen et al., 2005; Ishibashi et al., 1997). The diversity of lactic acid bacteria colonizing the human digestive system is high; however, the species composition is constantly changing as most of the species colonize the gastrointestinal tract for only a short period (Korhonen, 2010). Microorganisms in the adult intestine outnumber by 10-fold cells constituting the human body. The microbial composition for each individual is unique, depending on age, diet, diseases and environmental factors (Qin J. et al., 2010). LAB have been widely used as probiotic bacteria in the human gastrointestinal tract, contributing to pathogen inhibition and immunomodulation (Zhang et al., 2011).

The natural occurrence of lactic acid bacteria on plants (fruits, vegetables and grains) as well as in milk permitted their use in biotechnology (Makarova et al., 2006). *Lactobacillus, Pediococcus, Leuconostoc* and *Oenococcus* which reside on grapes, enable fruit fermentation and wine production (de Nadra, 2007). Also, LAB can occur naturally or be intentionally added as starter cultures during plant, meat and dairy fermentation (Korhonen, 2010).

In marine environments LAB play a role in the breakdown of organic matter. In the last decade LAB belonging to the following genera: *Amphibacillus*, *Alkalibacterium*, *Marinilactibacillus*, *Paraliobacillus*, *Halolactibacillus* were isolated from the samples taken from the sea and oceanic as well as from animals that inhabit these ecosystems. These bacteria were named "marine LAB" (Ishikawa et al., 2005).

### **3.2. Lactic acid bacteria – Influence on hydrogen producers**

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

pyruvate formate lyase, FHL – formate hydrogen lyase.

hydrogen production is shown in red.

(de Vos et al., 2009).

bacteria is genus *Lactobacillus* which comprises over 145 species. Genera *Bifidobacterium* and *Propionibacterium* (class: *Actinobacteria*) as well as spore forming rods belonging to the order *Bacillales*, family *Sporolactobacillaceae*, genus *Sporolactobacillus* constitute further groups of LAB. With the exception of bacteria belonging to the genera *Lactobacillus*, *Carnabacterium*, *Weissella* and *Sporolactobacillus* which are rods, other species of lactic acid bacteria are cocci

LdhA – lactate dehydrogenase, PoxB – pyruvate oxydase, PTA – phosphotransacetylase, ACK – acetate kinase, PFL –

LAB are microorganisms ubiquitous in the environment. Due to their high nutritional requirements, they are usually found in environments rich in carbohydrates, amino acids and nucleotides. On the other hand, they show considerable adaptation to the harsh

The digestive tracts of man and animals are among the environments where LAB occur. They have been reported in saliva, the small intestine and colon (Korhonen, 2010). The development of the gastrointestinal microflora in infants is influenced by contact with diverse microflora of the mother and of the closest surrounding. The main species found in both infants and adults are *Lactobacillus ruminis, L. salivarius, L. gasseri*, *L. reuteri* as well as *Bifidobacterium longum* and *B. breve* (Salminen et al., 2005; Ishibashi et al., 1997). The diversity

**Figure 2.** The scheme of mixed-acid fermentation (*Escherichia coli*-type). The pathway leading to

conditions, which allows them to inhabit a range of various niches (Korhonen, 2010).

Interestingly, lactic acid bacteria are often detected in mesophilic hydrogen-producing consortia as bacteria that accompany hydrogen producers. The technique most commonly used for analyzing the diversity of hydrogen-producing microbial communities is polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), followed by either direct sequencing or cloning and sequencing of DGGE bands. One of the disadvantages of this method is underestimation of the true bacterial diversity due to the fact that only the most prominent DGGE bands are analyzed. Various studies have shown that DGGE bands representing LAB are one of the most dominant bands (Fang et al., 2002; Kim et al., 2006; Li et al., 2006; Wu et al., 2006; Hung et al., 2007; Ren et al., 2007; Jo et al., 2007; Lo et al., 2008; Sreela-or et al., 2011). Another method of analyzing the biodiversity of hydrogen-producing consortia is cloning and sequencing of the 16S rDNA gene amplified on the total DNA isolated from the culture probes. Also with this method, sequences related to lactic acid bacteria have been detected (Yang et al., 2007). An alternative method used by our group for the first time to perform metagenomic analysis of hydrogen-producing microbial communities is 454-pyrosequencing. Our results showed that *Clostridiaceae, Enterobacteriaceae* and heterolactic fermentation bacteria, mainly *Leuconostocaeae,* were the most dominant bacteria in hydrogen-producing consortia under optimal condition for gas production (Chojnacka et al., 2011).

The aim of the chapter is a provocative discussion on the true role of LAB in hydrogenproducing bioreactors and their influence on hydrogen producers. Table 1 presents a set of selected studies which examine the possible influence of lactic acid bacteria on hydrogen production during dark fermentation.


Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 495

**References** 

Fang et al., 2002

Wu et al., 2006

Li et al., 2006

Lo et al., 2008

**Subject of examination Results and suggested influence of LAB** 

**discussed** 

discussed.

discussed.

Fermentative hydrogen production from sucrosecontaining wastewater in a well-mixed reactor and DGGE

analysis of bacterial

granular sludge.

community structure of the

Fermentative hydrogen production from sucrose in a continuously stirred anaerobic

bioreactor seeded with silicone-immobilized sludge and DGGE analysis of bacterial community structure of the

Fermentative hydrogen production from sucrose in a continuous stirred tank reactor and DGGE analysis of bacterial community structure of the

Fermentative hydrogen production from sucrose or xylose in a continuous dark fermentation bioreactor and DGGE analysis of the bacterial

community structure.

granular sludge.

granular sludge.

**on hydrogen producers** 

*Clostridium* species (*C. pasteurianum, C.* 

*Sporolactobacillus racemicus* were detected in the bioreactor. A high-rate fermentative hydrogen production was observed. The role of LAB (*Sporolactobacillus racemicus*) in the microbial community is not discussed.

DGGE analysis revealed the presence of representatives of the following genera and species: *Clostridium* (*C. intestinale* and *C. pasteurianum*), *Escherichia coli*, *Streptococcus*  sp., *Klebsiella pneumoniae.* A high-rate fermentative hydrogen production was observed. The role of LAB (*Streptococcus*  sp.) in the microbial community is not

*Clostridium cellulosi, Clostridium* sp., *Klebsiella ornithinolytica, Prevotella* sp. and *Leuconostoc pseudomesenteroides* were detected in the bioreactor. A high-rate fermentative hydrogen production was

observed. The role of LAB (*L. pseudomesenteroides*) in the microbial hydrogen-producing community is not

*Clostridium* species (*C. butyricum, C. pasteurianum* on sucrose and *C. celerecrescens* on xylose), *Klebsiella pneumoniae, K. oxytoca, Streptococcus* sp., *Escherichia* sp., *Pseudomonas* sp. *Dialister* sp.,

*Bacillus* sp., *Bifidobacterium* sp. were

detected in the bioreactor. The role of LAB (*Streptococcus* sp. and *Bifidobacterium* sp.) in the microbial community is not discussed.

*tyrobutyricum, C. acidisoli*) and

**B Role of LAB in hydrogen-producing consortia not** 

**A. Negative role of LAB**

Investigation of the effects of LAB on hydrogen fermentation of bean curd manufacturing waste in a series of co-cultures of *Clostridium butyricum* and two strains of *C. acetobutylicum*  with *Lactobacillus paracasei* and

*Enterococcus durans*.

analysis of bacterial community structure.

DGGE examination of

continuous culture.

Investigation of hydrogen production from food waste in

anaerobic mixed cultures and DGGE analysis of microbial

batch fermentation by

community.

microbial community during unstable hydrogen production from food waste of kimchi in a

Fermentative hydrogen production from molasses in continuous stirred-tank reactors and DG-DGGE (double gradient denaturating gradient gel electrophoresis)

**Subject of examination Results and suggested influence of LAB** 

bacteriocins.

production).

competition.

production).

**on hydrogen producers** 

Inhibition of hydrogen producers by LAB

(replacement of hydrogen fermentation by lactic acid fermentation); (ii) excretion of

*Desulfovibrio ferrireducens, Actinomyces* sp., *Klebsiella oxytoca*, *Acidovorax* sp., uncultured *Actinobacterium* and *Bacteroidetes* were detected in the bioreactor where the main non-gaseous end products were ethanol, butyric acid and acetic acid. Negative role

due to (i) substrate competition

*C. pasteurianum, Lactococcus* sp.,

of *Lactococcus* species: inhibition of hydrogen production by substrate competition (competitive ethanol

Conversion of hydrogen fermentation to lactic acid fermentation due to shifts in the microbial community structure from *Clostridium* spp. to *Lactobacillus* spp. Negative role of LAB: substrate

*Clostridium* species (*C. butyricum, C. acetobutylicum, C. beijerinckii, Clostridium*  sp.) were the dominant hydrogen producers. Negative role of LAB representatives (*Lactobacillus* sp.,

*Enterococcus* sp.): inhibition of hydrogen production by substrate competition (competitive ethanol and lactic acid

**References** 

Noike et al.,

Ren et al., 2007

Jo et al., 2007

Sreela-or et al., 2011

2002



Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 497

other bacteria. These observations derive from examinations of both batch (Sreela-or et al., 2011) and continuous (Ren et al., 2007; Jo et al., 2007) mixed cultures as well as co-cultures where one component was a representative of clostridia and the second one of lactic acid bacteria (Noike et al., 2002). Heat treatment was proposed as a method of eliminating lactic

Substrate competition includes changes in the type of fermentation occurring in the bioreactors during long-term continuous processes and replacement of hydrogen fermentation by lactic acid or ethanol fermentation (Noike et al., 2002; Jo et al., 2007; Ren et al., 2007; Sreela-or et al., 2011). In all of the studies decrease in hydrogen production was observed with simultaneous increase of lactic acid and ethanol concentrations in the

The hypothesis that bacteriocins may act as inhibitors of hydrogen production was postulated by Noike and co-workers (2002), who showed in a series of co-cultures experiments that cessation of hydrogen production by *C. acetobutylicum* and *C. butyricum* was caused by both the presence of *Enterococcus durans* and *Lactobacillus paracasei* as well as supernatants from their culture media. Moreover, treatment of the supernatants with

Studies listed in part B of Table 1 determined the presence of lactic acid bacteria in hydrogenproducing consortia; yet, their role in these microbial communities is not discussed. It is noteworthy that (i) those papers discuss efficient systems of biohydrogen production and (ii) studies were performed under optimal conditions for hydrogen production (Fang et al., 2002;

Part C of Table 1 presents the only so far available studies arguing that LAB could play a positive role in hydrogen-producing microbial communities and stimulate hydrogen

Hung and colleagues (2007) studied the efficiency of fermentative hydrogen production from glucose in anaerobic agitated granular sludge bed bioreactors under different substrate concentration and hydraulic retention times (HRT). PCR-DGGE and FISH methods were used to analyze the biohydrogen-producing microbial community of the granular sludge. The bacterial community was composed of *Clostridium* sp. (possibly *C. pasteurianum*), *Klebsiella oxytoca* and *Streptococcus* sp. The percentage of *Streptococcus* sp. contributing to the microbial community was dependent on the HRT. The shorter HRT, meaning the faster the flow of the medium and increased dilution rate, the higher the contribution of *Streptococcus*  sp. in the bacterial consortium was observed. Formation of granular sludge enables biomass retention. FISH analysis revealed that *Streptococcus* cells are located inside granules surrounded by *Clostridium* cells. Authors postulate that *Streptococcus* cells may act as the

According to Yang et al. (2007) some LAB are able to produce hydrogen. They declare isolation of strains from the genus *Lactobacillus* capable of hydrogen production during

trypsin recovered normal hydrogen production by selected clostridial strains.

Kim et al., 2006; Li et al., 2006; Wu et al., 2006; Hung et al., 2007).

acid bacteria (Noike et al., 2002; Baghchehsaraee et al., 2008).

effluents or fluid phase of the culture.

seed for sludge granule formation.

lactose fermentation.

production.

**Table 1.** A set of selected studies demonstrating the contribution of LAB in hydrogen-producing cultures and presenting their possible influence on hydrogen production.

Some studies argue that development of LAB in bioreactors may inhibit hydrogen production (Table 1, part A). Cessation of hydrogen generation by LAB was suggested to be due to (i) substrate competition and/or (ii) excretion of bacteriocins inhibiting growth of other bacteria. These observations derive from examinations of both batch (Sreela-or et al., 2011) and continuous (Ren et al., 2007; Jo et al., 2007) mixed cultures as well as co-cultures where one component was a representative of clostridia and the second one of lactic acid bacteria (Noike et al., 2002). Heat treatment was proposed as a method of eliminating lactic acid bacteria (Noike et al., 2002; Baghchehsaraee et al., 2008).

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

**C. Positive role of LAB** 

Fermentative hydrogen production from glucose in anaerobic agitated granular sludge bed bioreactors and DGGE and FISH analyses of

the granular sludge.

Fermentative hydrogen production from cheese whey

wastewater by mixed continuous cultures and molecular analysis of the consortium by cloning and sequencing of the 16S rDNA gene amplified on the total DNA isolated from the culture

Fermentative hydrogen production from molasses in packed bed bioreactors and metagenomic analysis of bacterial biofilms and granules by 454-pyrosequencing.

probe.

**Subject of examination Results and suggested influence of LAB** 

**on hydrogen producers** 

The DGGE analysis showed that the

bacterial community was mainly composed of *Clostridium* sp., *Klebsiella oxytoca* and *Streptococcus* sp. A high-rate fermentative hydrogen production was observed. The FISH images suggested that *Streptococcus*  cells acted as seeds for granule formation.

The most prevalent bacteria, representing approximately 50% of the total sequences analyzed, were representatives of the genus *Lactobacillus*. Remaining sequences

belonged to the genera *Olsenella, Clostridium* and *Prevotella*. Decrease in hydrogen production was accompanied by the reductions in the number of detected bacteria from the genus *Lactobacillus*. Authors declare isolation of *Lactobacillus* bacteria capable of hydrogen production in

the process of lactose fermentation.

Metagenomic analysis of microbial consortia by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the most dominant bacteria were the

representatives of the *Firmicutes*  (*Clostridiaceae* and *Leuconostocaeae*) and *Gammaproteobacteria* (*Enterobacteriaceae*)*.*  Bacteria of heterolactic fermentation were one of the predominant microbes in hydrogen-producing consortia. The speculation that LAB may favor hydrogen production is discussed. For details see Tables 2-4, Figures 3-5 and description in

the text.

cultures and presenting their possible influence on hydrogen production.

**Table 1.** A set of selected studies demonstrating the contribution of LAB in hydrogen-producing

Some studies argue that development of LAB in bioreactors may inhibit hydrogen production (Table 1, part A). Cessation of hydrogen generation by LAB was suggested to be due to (i) substrate competition and/or (ii) excretion of bacteriocins inhibiting growth of

**References** 

Hung et al.,

Yang et al., 2007

Chojnacka et al., 2011

2007

Substrate competition includes changes in the type of fermentation occurring in the bioreactors during long-term continuous processes and replacement of hydrogen fermentation by lactic acid or ethanol fermentation (Noike et al., 2002; Jo et al., 2007; Ren et al., 2007; Sreela-or et al., 2011). In all of the studies decrease in hydrogen production was observed with simultaneous increase of lactic acid and ethanol concentrations in the effluents or fluid phase of the culture.

The hypothesis that bacteriocins may act as inhibitors of hydrogen production was postulated by Noike and co-workers (2002), who showed in a series of co-cultures experiments that cessation of hydrogen production by *C. acetobutylicum* and *C. butyricum* was caused by both the presence of *Enterococcus durans* and *Lactobacillus paracasei* as well as supernatants from their culture media. Moreover, treatment of the supernatants with trypsin recovered normal hydrogen production by selected clostridial strains.

Studies listed in part B of Table 1 determined the presence of lactic acid bacteria in hydrogenproducing consortia; yet, their role in these microbial communities is not discussed. It is noteworthy that (i) those papers discuss efficient systems of biohydrogen production and (ii) studies were performed under optimal conditions for hydrogen production (Fang et al., 2002; Kim et al., 2006; Li et al., 2006; Wu et al., 2006; Hung et al., 2007).

Part C of Table 1 presents the only so far available studies arguing that LAB could play a positive role in hydrogen-producing microbial communities and stimulate hydrogen production.

Hung and colleagues (2007) studied the efficiency of fermentative hydrogen production from glucose in anaerobic agitated granular sludge bed bioreactors under different substrate concentration and hydraulic retention times (HRT). PCR-DGGE and FISH methods were used to analyze the biohydrogen-producing microbial community of the granular sludge. The bacterial community was composed of *Clostridium* sp. (possibly *C. pasteurianum*), *Klebsiella oxytoca* and *Streptococcus* sp. The percentage of *Streptococcus* sp. contributing to the microbial community was dependent on the HRT. The shorter HRT, meaning the faster the flow of the medium and increased dilution rate, the higher the contribution of *Streptococcus*  sp. in the bacterial consortium was observed. Formation of granular sludge enables biomass retention. FISH analysis revealed that *Streptococcus* cells are located inside granules surrounded by *Clostridium* cells. Authors postulate that *Streptococcus* cells may act as the seed for sludge granule formation.

According to Yang et al. (2007) some LAB are able to produce hydrogen. They declare isolation of strains from the genus *Lactobacillus* capable of hydrogen production during lactose fermentation.
