**3.3. Fermentative hydrogen production and microbial analysis of bacterial biofilms and granular sludge formed in packed bed bioreactors**

We developed an effective system of bacterial hydrogen production based on long-term continuous cultures (from an inoculum of a lake bottom sediment) grown on sugar beet molasses in packed bed reactors filled with granitic stones (Chojnacka et al., 2011). In separate cultures, two consortia of anaerobic fermentative bacteria producing hydrogen-rich gas developed on the stones as biofilms. Furthermore, in one of the cultures a granular sludge was also observed (Figures 3 and 4). Cultures were named, respectively, (i) the culture with stone biofilm only and (ii) granular sludge culture. Both cultures were regularly renewed by removal of an excess of biomass.

Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 499

**Figure 4.** Scanning electron micrographs of structures formed by selected consortia of fermentative bacteria grown on M9 medium containing molasses: (a – c) granules; (d – f) bacterial biofilm formed on

the granitic stones filling the bioreactor in the granular sludge culture.

Analysis of the surface topography of biofilms from both cultures revealed their porous, irregular structure with many cavities and channels. Bacteria appeared to be suspended in and surrounded by a matrix substance. The granules were white and light cream in color, with a diameter between 0.2 – 2 mm, and of hard structure, resistant to squashing or crumbling. Moreover, the granules were clustered in structures resembling bunches of grapes with a noticeable net of channels. Similar to the bacterial biofilm, the granules consisted of bacterial cells surrounded by a matrix.

**Figure 3.** Images of the two structures formed by selected consortia of fermentative bacteria grown in a bioreactor on M9 medium containing molasses: (a) stones covered with bacterial biofilm (b) the granular sludge.

Metagenomic analysis of microbial communities by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the overall biodiversity of hydrogen-producing cultures was quite small. Stone biofilm from the culture without the granular sludge was dominated by *Clostridiaceae* and heterolactic fermentation bacteria, mainly *Leuconostocaeae*. Representatives of *Leuconostocaeae* and *Enterobacteriaceae* were dominant in both the granules and the stone biofilm formed in the granular sludge culture. The granular sludge contained bacteria of heterolactic fermentation, dominated by *Leuconostoc* species as well as unclassified *Streptococcaceae* and unclassified *Enterobacteriaceae*. Surprisingly, sequences representing the *Clostridiaceae* were in a relative minority (Table 2).

renewed by removal of an excess of biomass.

consisted of bacterial cells surrounded by a matrix.

*Clostridiaceae* were in a relative minority (Table 2).

granular sludge.

**3.3. Fermentative hydrogen production and microbial analysis of bacterial** 

We developed an effective system of bacterial hydrogen production based on long-term continuous cultures (from an inoculum of a lake bottom sediment) grown on sugar beet molasses in packed bed reactors filled with granitic stones (Chojnacka et al., 2011). In separate cultures, two consortia of anaerobic fermentative bacteria producing hydrogen-rich gas developed on the stones as biofilms. Furthermore, in one of the cultures a granular sludge was also observed (Figures 3 and 4). Cultures were named, respectively, (i) the culture with stone biofilm only and (ii) granular sludge culture. Both cultures were regularly

Analysis of the surface topography of biofilms from both cultures revealed their porous, irregular structure with many cavities and channels. Bacteria appeared to be suspended in and surrounded by a matrix substance. The granules were white and light cream in color, with a diameter between 0.2 – 2 mm, and of hard structure, resistant to squashing or crumbling. Moreover, the granules were clustered in structures resembling bunches of grapes with a noticeable net of channels. Similar to the bacterial biofilm, the granules

**Figure 3.** Images of the two structures formed by selected consortia of fermentative bacteria grown in a bioreactor on M9 medium containing molasses: (a) stones covered with bacterial biofilm (b) the

(a) (b)

Metagenomic analysis of microbial communities by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the overall biodiversity of hydrogen-producing cultures was quite small. Stone biofilm from the culture without the granular sludge was dominated by *Clostridiaceae* and heterolactic fermentation bacteria, mainly *Leuconostocaeae*. Representatives of *Leuconostocaeae* and *Enterobacteriaceae* were dominant in both the granules and the stone biofilm formed in the granular sludge culture. The granular sludge contained bacteria of heterolactic fermentation, dominated by *Leuconostoc* species as well as unclassified *Streptococcaceae* and unclassified *Enterobacteriaceae*. Surprisingly, sequences representing the

**biofilms and granular sludge formed in packed bed bioreactors** 

**Figure 4.** Scanning electron micrographs of structures formed by selected consortia of fermentative bacteria grown on M9 medium containing molasses: (a – c) granules; (d – f) bacterial biofilm formed on the granitic stones filling the bioreactor in the granular sludge culture.


Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 501

Results of the metagenomic analysis by 454-pyrosequencing were confirmed by FISH (Fluorescence-In-Situ-Hybridization) analysis (Fig. 5) as well as by isolatating of lactic acid

Both, the stone biofilm and granules are composed of bacteria of many different shapes. As judged from fluorescence *in situ* hybridization, the relative abundance of selected bacterial groups varied during the rounds of bioreactor cycles. At the very beginning of biofilm development clostridial and lactobacilli cells were detected only sporadically among gammaproteobacteria (Fig. 5A a-c). In the growing biofilm systematic increase of Firmicutes

bacteria from the culture (Table 3).

(especially lactobacilli) cells was observed (Fig. 5Bd-e).

A: young biofilm; a-clostridia/dyLight405, b-lactobacilli/TAMRA; c-gammaproteobacteria/CY3, B: mature biofilm; d-firmicutes/CY5; e-lactobacilli/TAMRA; d', e'-in combination with phase contrast.

stained with fluorescently labeled specific probes.

outnumbered homofermentatives.

**Figure 5.** FISH image of the hydrogen-producing biofilm from the granular sludge culture described previously (Chojnacka et al., 2011) analyzed by confocal laser fluorescence microscopy. The sample was

A cultivable approach with the use of media promoting the growth of lactic acid bacteria (MRS, M17) revealed that the bioreactor was inhabited by a vast number of these bacteria. Similarly to the metagenomic data, the majority of growing colonies represented *Leuconostoc* or *Lactobacillus* genera. All in all, six different species listed in Tab. 3 were isolated. It was determined that heterofermentative species (*Leuconostoc*, *L. brevis*, *L. rhamnosus*) slightly

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

B – stone biofilm from the culture without granular sludge; Bg – stone biofilm from the granular sludge culture; G – granules from the granular sludge culture.

**Table 2.** Number of reads assigned to respective taxonomic branches of 16S rRNA gene fragments amplified from the total DNA pool from bacterial communities formed in bioreactors.

Results of the metagenomic analysis by 454-pyrosequencing were confirmed by FISH (Fluorescence-In-Situ-Hybridization) analysis (Fig. 5) as well as by isolatating of lactic acid bacteria from the culture (Table 3).

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

B – stone biofilm from the culture without granular sludge; Bg – stone biofilm from the granular sludge culture; G –

**Table 2.** Number of reads assigned to respective taxonomic branches of 16S rRNA gene fragments

amplified from the total DNA pool from bacterial communities formed in bioreactors.

granules from the granular sludge culture.

Both, the stone biofilm and granules are composed of bacteria of many different shapes. As judged from fluorescence *in situ* hybridization, the relative abundance of selected bacterial groups varied during the rounds of bioreactor cycles. At the very beginning of biofilm development clostridial and lactobacilli cells were detected only sporadically among gammaproteobacteria (Fig. 5A a-c). In the growing biofilm systematic increase of Firmicutes (especially lactobacilli) cells was observed (Fig. 5Bd-e).

A: young biofilm; a-clostridia/dyLight405, b-lactobacilli/TAMRA; c-gammaproteobacteria/CY3, B: mature biofilm; d-firmicutes/CY5; e-lactobacilli/TAMRA; d', e'-in combination with phase contrast.

**Figure 5.** FISH image of the hydrogen-producing biofilm from the granular sludge culture described previously (Chojnacka et al., 2011) analyzed by confocal laser fluorescence microscopy. The sample was stained with fluorescently labeled specific probes.

A cultivable approach with the use of media promoting the growth of lactic acid bacteria (MRS, M17) revealed that the bioreactor was inhabited by a vast number of these bacteria. Similarly to the metagenomic data, the majority of growing colonies represented *Leuconostoc* or *Lactobacillus* genera. All in all, six different species listed in Tab. 3 were isolated. It was determined that heterofermentative species (*Leuconostoc*, *L. brevis*, *L. rhamnosus*) slightly outnumbered homofermentatives.


Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence? 503

**granular sludge** 

**Culture containing granular sludge** 

cultures containing only biofilm. The formic and acetic acids present in the medium were utilized by both cultures. It is noteworthy that in the granular sludge culture rich in heterolactic bacteria showing very good performance in hydrogen production and a high content of butyric acid, the number of *Clostridiales* sequences was significantly lower than in

Based on our results presented in the study of Chojnacka et al. (2011) we speculate that LAB may possibly play a significant but not fully understood and perhaps underestimated role in the hydrogen producing communities. This hypothesis is based on two observations: (i) the higher the number of LAB in the hydrogen-producing community, the more efficiently hydrogen is produced; (ii) complete consumption of lactic acid, significantly increased concentration of butyric acid as well as larger hydrogen yield in the culture containing

the biofilm-only culture.

granular sludge than in that with just the biofilm.

**Total gas production (cm3/min/working volume of** 

Others (NH3, H2S, formic, acetic, propionic and

**Hydrogen production (cm3/day/working volume of** 

**Net production of the non-gaseous end products** 

for hydrogen production based on the study of Chojnacka et al. (2011).

**(mg/L):** 

**Parameter Culture without** 

**Composition of fermentation gas (%):**

**the bioreactor) 6.6 9.5** 

Hydrogen 35.7 % 48.6 % Carbon dioxide 60% 47.1 % Water vapor ~4.3% ~4.3 % Methane 0.0004 % 0.0004%

butyric acids) ~1% ~1%

**the bioreactor) 3393 6649** 

**Yield of hydrogen (moles H2/mole of sucrose) 2.8 5.43** 

Lactic acid 2419 ± 42.6 0 Formic acid 0 0 Acetic acid 0 0 Propionic acid 248 ± 0.7 0 Butyric acid 4331 ± 60.0 7641 ± 33.1 Ethanol (%) 0.06 ± 0.002 0.1 ± 0.004

**Table 4.** Parameters describing two cultures of hydrogen-producing bacteria under optimal conditions

**Table 3.** The species of LAB isolated from the hydrogen-producing bioreactor.

Samples were collected from both, stone (biofilm) and liquid phase of hydrogen-producing culture, and plated on selective media for lactic acid bacteria. Plates were incubated under anaerobic conditions. Obtained colonies were tested for Gram positivity and lack of catalase enzyme. For strains which gave positive results, the V3 fragment of the 16S rRNA gene was amplified. Subsequently, fragments were analyzed using MSSCP technique. Strains with unique or representative gel patterns were chosen for further studies based on amplification and sequencing of 16S rRNA gene. Resulting sequences were identified by comparison to known sequences using the NCBI database. Names of homofermentative species are written in bold.

Formation of granular sludge rich in heterolactic bacteria significantly enhanced hydrogen production. Table 4 presents a list of parameters describing and comparing the two bacterial cultures that were the subject of the study of Chojnacka et al. (2011), under optimal conditions for hydrogen production. Significantly higher total gas production was observed for the culture containing granular sludge than for the biofilm-only culture (9.5 vs. 6.6 cm3/min/working volume of the bioreactor). Furthermore, the percentage contribution of hydrogen was almost 49 and 36 %, whereas of carbon dioxide 47 and 60%, in the former and latter cultures, respectively. The granular sludge culture produced hydrogen at the rate of 6649 cm3/day/working volume of the bioreactor, whereas the biofilm-only culture at the rate of 3393 cm3/day/working volume of the bioreactor. Fermentation gas produced by both cultures contained 0.0004% methane, meaning that it was practically methane-free. Consequently, under optimal conditions, the culture containing granular sludge rich in heterolactic bacteria was two-fold more effective in producing hydrogen than that containing biofilm only: 5.43 moles of H2 vs. 2.8 moles of H2/mole of sucrose from molasses, respectively.

It is known that butyrate is the predominant metabolite during butyric acid fermentation at pH 5.0 – 5.5 (Li and Fang, 2007). The analysis of the non-gaseous fermentation products in both cultures in the study of Chojnacka et al. (2011) revealed that butyric acid was the main metabolite with partial contribution of ethanol. Concentration of butyric acid was almost 1.8-fold higher in the culture containing granular sludge than in the biofilm-only culture. No net production of lactic and propionic acids was observed in the granular sludge culture, whereas these were the second and third most abundant fermentation products in the cultures containing only biofilm. The formic and acetic acids present in the medium were utilized by both cultures. It is noteworthy that in the granular sludge culture rich in heterolactic bacteria showing very good performance in hydrogen production and a high content of butyric acid, the number of *Clostridiales* sequences was significantly lower than in the biofilm-only culture.

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

*Lactobacillus plantarum* 46,7%

*Enterococcus casseliflavus* 0,5% *Leuconostoc mesenteroides* 46,7% *Leuconostoc mesenteroides* ssp*. mesenteroides* 0,5% *Lactobacillus brevis* 5,1% *Lactobacillus rhamnosus* 0,5%

in bold.

respectively.

**Isolate Isolation ratio Homofermenters:heterofermenters ratio** 

Samples were collected from both, stone (biofilm) and liquid phase of hydrogen-producing culture, and plated on selective media for lactic acid bacteria. Plates were incubated under anaerobic conditions. Obtained colonies were tested for Gram positivity and lack of catalase enzyme. For strains which gave positive results, the V3 fragment of the 16S rRNA gene was amplified. Subsequently, fragments were analyzed using MSSCP technique. Strains with unique or representative gel patterns were chosen for further studies based on amplification and sequencing of 16S rRNA gene. Resulting sequences were identified by comparison to known sequences using the NCBI database. Names of homofermentative species are written

Formation of granular sludge rich in heterolactic bacteria significantly enhanced hydrogen production. Table 4 presents a list of parameters describing and comparing the two bacterial cultures that were the subject of the study of Chojnacka et al. (2011), under optimal conditions for hydrogen production. Significantly higher total gas production was observed for the culture containing granular sludge than for the biofilm-only culture (9.5 vs. 6.6 cm3/min/working volume of the bioreactor). Furthermore, the percentage contribution of hydrogen was almost 49 and 36 %, whereas of carbon dioxide 47 and 60%, in the former and latter cultures, respectively. The granular sludge culture produced hydrogen at the rate of 6649 cm3/day/working volume of the bioreactor, whereas the biofilm-only culture at the rate of 3393 cm3/day/working volume of the bioreactor. Fermentation gas produced by both cultures contained 0.0004% methane, meaning that it was practically methane-free. Consequently, under optimal conditions, the culture containing granular sludge rich in heterolactic bacteria was two-fold more effective in producing hydrogen than that containing biofilm only: 5.43 moles of H2 vs. 2.8 moles of H2/mole of sucrose from molasses,

It is known that butyrate is the predominant metabolite during butyric acid fermentation at pH 5.0 – 5.5 (Li and Fang, 2007). The analysis of the non-gaseous fermentation products in both cultures in the study of Chojnacka et al. (2011) revealed that butyric acid was the main metabolite with partial contribution of ethanol. Concentration of butyric acid was almost 1.8-fold higher in the culture containing granular sludge than in the biofilm-only culture. No net production of lactic and propionic acids was observed in the granular sludge culture, whereas these were the second and third most abundant fermentation products in the

**Table 3.** The species of LAB isolated from the hydrogen-producing bioreactor.

0,89

Based on our results presented in the study of Chojnacka et al. (2011) we speculate that LAB may possibly play a significant but not fully understood and perhaps underestimated role in the hydrogen producing communities. This hypothesis is based on two observations: (i) the higher the number of LAB in the hydrogen-producing community, the more efficiently hydrogen is produced; (ii) complete consumption of lactic acid, significantly increased concentration of butyric acid as well as larger hydrogen yield in the culture containing granular sludge than in that with just the biofilm.


**Table 4.** Parameters describing two cultures of hydrogen-producing bacteria under optimal conditions for hydrogen production based on the study of Chojnacka et al. (2011).
