Production of Bio-Hydrogen Gas and Other Metabolic Gases by Anaerobic Bacteria Grown on Molasses

The study had aimed to characterize the production of hydrogen gases by anaerobic bacteria. One isolate was found in sheep ruminal fluid and four isolates were obtained from the activated sludge. These isolates were identified by microscopic methods and by rRNA sequences. One ruminal bacterium was identified as Escherichia coli, and it was found that these isolates from activated sludge were related to Clostridium botulinum, C. perfringens and C. difficile. One strain could not be assigned to any species but was similar to C. botulinum. Growth and production of the metabolic gases with molasses as sole carbon source were measured during the anaerobic cultivation by Micro-Oxymax (Columbus Instruments, Columbus, OH, U.S.A.) gas analyzer. One of the most available saccharidic waste products is molasses. The growth on molasses as carbon source was done to test the production of H2. It was found that all tested Clostridium isolates (AK 1-4, AK 1-5, AK 1-9 and AK 1-12) and E. coli isolate (No 224) had utilized molasses as carbon source monitored by production of CO2 gas. All these strains produced H2 gas, and CO gas in concentration range 102 μmol L –1 , and H2S gas in concentrations lower by one order of magnitude. Kinetics of evolution of these gases was different suggesting that they are produced by independent processes. Results show that metabolic gases are produced mainly in the exponential phase of growth.


Introduction
Hydrogen gas (H 2 ) is considered the promising clean fuel [1], due to its potentially higher efficiency of conversion to usable power, low or none pollutant formation and high energy density [2]. Consequently, low-cost and sufficient supply of H 2 could soon become in urgent demand [3]. Hydrogen as fuel can be produced by biological or nonbiological processes. It can be produced using fermentative bacteria, photosynthetic bacteria or algae. Biological H 2 production using fermentative, photosynthetic bacteria, or algae is an environmentally friend and energy saving process [4] [5]. Therefore, it is a feasible alternative for global H 2 supply in the future [6].
The biological production of microbial H 2 as fuel can be classified cyanobacteria; photodecomposition of organic compounds by photosynthetic bacteria (photo-fermentation); and dark fermentation by fermentative bacteria [7]. Production of Fermentative H 2 has good prospects for the biotechnological applications, [8] [9]. In the dark fermentation process, different microbes can be used anaerobically to breakdown carbohydrate-rich substrates to H 2 , organic acids (e.g., lactic, acetic, butyric, etc.) and alcohols [8] [10] [11]. Among bacteria which are used for the dark fermentation, clostridia play an important role. Their robust includes diverged fermentative metabolism, and easy isolation of clostridial strains, stability of cultures that includes spore formation, and presence of hydrogenases [12] favors clostridia as potential H 2 producers [13]. The suitability of Clostridium spp. for H 2 production was confirmed by more studies [14] [15]. More waste materials were used for H 2 production in the biotechnological applications. Many literatures described production of H 2 from municipal wastes, [16] used apple pomace and [17] treated sugarcane bagasse by Clostridium butylicum. However, less complex substrates, such as cellulose could be degraded by cellulolytic clostridia [18] [19]. Several studies were made to increase the production of H 2 by using cocultures, cellulolytic and non-cellulolytic strains, in order to increase cellulose hydrolysis [19]. Similar approach adopted [20]. Co-cultured Clostridium butylicum with Enterococcus saccharolyticum in order to improve hydrolysis of cellobiose, or [4], Succeeded to co-culture Bacillus thermoamylovorans and several Clostridium species in order to improve hydrolytic processes; also co-cultures of Clostridium butyricum and Rhodobacter sphaeroides were successfully tested as a model of combined phototrophic and dark fermentation process of H 2 production [21] [22]. Application used co-culture of C. butyricum with facultative anaerobe Citrobacter freundii on under anaerobic condition, which could damage physiological processes in clostridia. Several attempts, Appeared to use genetics for improving efficiency of H 2 production. Heterologous expression of clostridial hydrogenase in Escherichia coli augmented H 2 production [23]. On the other hand, autologous overexpression of hydrogenase in C. acetobutylicum did not augment the H 2 production [24]. Molasses are alternative carbohydrate rich substrate, and are well source of sucrose. It was used as a sole carbon source for H 2 gas evolution [25]. Some study testing molasses for hydrogen production, found that the high hydrogen yield was always associated with a high level of ethanol production [26].
The hydrogen production by the dark fermentation of molasses from sugar industry [28].
Other study used the sugar beet molasses to hydrogen and methane production as a result of two-stage anaerobic digestion [29].
Also they tested molasses for hydrogen and methane production [30].

Isolation of the Bacterial Strains
The sheep ruminal fluid and the activated sludge were used to isolate the bacterial culture. For pure colonies of bacteria, a sample from the ruminal fluid had been taken from the sheep stomach or from the activated sludge, and transferred into physiological solution (supplemented with 0.02% Tween 80) and serially diluted up to 10 6 times under anaerobic conditions. Then the inoculated a physiological solution was vaccinated under anaerobic conditions on the solid reinforced clostridial medium (RCM), which was equipped on the Petri dishes in order to obtain pure and individually colonies of anaerobic bacteria. The bacteria were cultivated at 37°C in anaerobic cultivation chambers in the anaerobic atmosphere formed by BBL Gas Pack System (Becton Dickinson). All operations were performed in the Bactron I anaerobic chamber (Sheldon Laboratories).

Growth Curve Measurements
Single bacterial culture was cultivated in 5 ml media (RCM) and inoculated under anaerobic condition by bacterial species to have concentration of cells about 2.107ml -1 , and incubated under anaerobic conditions at 37°C with rotation (IKA KS 40000 ic control) (181 rpm). Growth of bacteria was monitored by measurement of A550 (Biochrom colorimeter Libra 52, U. K.) at 0 hour, and after every 4th hour until stationary phase was reached.

Molecular Taxonomy of Bacterial Isolates
Bacterial taxonomy was performed by colony PCR amplifying the 16S -23S ribosomal bacterial spacer. Fifty 50 µl PCR mix contains 1 µl 10 mM dNTP mix, 1.5 µl primer forward (B16S2 FWD 5´TTGTACACACCGCCCGTC´3.), 1.5 µl primer reverse *(B28S10 REW) (5´CCWTTCCCTCACGGTACT3´), 5 µl 10x Hot Master TM Tag buffer with Mg 2+ , 0.5 µl Hot Master TM Tag DNA polymerase, and 40.5 µl DNAase -free water. DNA was provided by transferring and a very small part of bacterial colonies with toothpick. PCR was started without the adding of the DNA polymerase in the thermocycler (Master Cycler personal Eppendorf, Germany), and after 15 minutes the DNA polymerase was added to the PCR mix. PCR products were purified and directly sequenced. Sequences were analyzed by BLAST.

Measurements of Metabolic Gases
Tested samples were prepared from pure bacterial cultures identified by 16S -23S ribosomal bacterial spacer DNA in 40 ml of liquid medium into 50 ml flasks with stoppers with inlet and outlet tubings and placed in Micro-Oxymax (Columbus Instruments, Columbus, OH, U.S.A.) gas analyzer equipped with sensors for O 2 , CO, CO 2 , H 2 S, H 2 and CH 4 from which traces of O 2 were removed by flushing with pyrogallol-treated N 2. Measurement started after the system was calibrated with calibration gases. Measurements were carried out at 37°C.

Results and Discussion
The evolution of CO 2 in tested strains was different and varied between 0.25 to 2.1% at the production maximum suggesting that not all isolates grow equally on this substrate. Differences were observed also in the production of H 2 S. Only Clostridium spp. (AK 1-12) produced H 2 S concomitantly with CO 2 (Figure 1). Also, CO gas was lasted during the exponential phase and it was not evolved concomitantly with CO 2 or H 2 S gas ( Figure 1). The evolution of gases by AK 1-4 in exponential phase of growth was not accompanied by the large H 2 gas production although the production of CO 2 gas was extensive and temporary ( Figure  2); and the evulation of CO 2 and H 2 gases of were lasted during the exponential phase of growth; but H 2 gas was consumed during the late (stationary) phase of cultivation, see (figure 2) which it is also shown that the evolution of H 2 S gas is not parallel with the evolution of H 2 furthermore CO gas was formed during the exponential phase in parallel with CO 2 gas.
In Clostridium AK 1-5 and E. coli isolates (Figure 3 and 4, respectively), H 2 and CO 2 gases were produced in the exponential phase of growth and accompanied by the evolution of small amount of H 2 S gas in AK 1-5 but not in E. coli (Figure 4).
In AK 1-5 isolate (Figure 3), CO gas was produced during exponential phase, and its production was not parallel with any one of produced gases. In AK 1-9 isolate (Figure 5), CO gas was produced and consumed during exponential phase and then produced again during the stationary phase of cultivation; in the Figure it is also shown that the evolution of CO gas is not parallel with the of CO 2 , H 2 or H 2 S.

Figure 1. Evolutions of Metabolic Gases on Molasses as Substrate by Clostridium Spp. (AK 1-12 Isolate).
Graphs were plotted from data produced by gas analyzer for O 2 , CO, H 2 , CO 2 and H 2 S. They illustrate three possible outputs of instrument from left to right: Rate of gas changes, gas accumulation and immediate gas concentration in%. Parameters in the linear scale, O 2 values omitted.   Graphs were plotted from data produced by gas analyzer for O 2 , CO, H 2 , CO 2 and H 2 S. They illustrate three possible outputs of instrument from left to right: Rate of gas changes, gas accumulation and immediate gas concentration in%. Parameters in the linear scale, O 2 values omitted.

Figure 3. Evolutions of Metabolic Gases on Molasses as Substrate by Clostridium Difficile (AK 1-5 Isolate).
Graphs were plotted from data produced by gas analyzer for O 2 , CO, H 2 , CO 2 and H 2 S. They illustrate three possible outputs of instrument from left to right: Rate of gas changes, gas accumulation and immediate gas concentration in%. Parameters in the linear scale, O 2 values omitted.

Figure 4. Evolutions of Metabolic Gases on Molasses as Substrate by E. Coli (No 2-24 Isolate).
Graphs were plotted from data produced by gas analyzer for O 2 , CO, H 2 , CO 2 and H 2 S. They illustrate three possible outputs of instrument from left to right: Rate of gas changes, gas accumulation and immediate gas concentration in%. Parameters in the linear scale, O 2 values omitted.

Figure 5. Evolutions of Metabolic Gases on Molasses as Substrate by Clostridium Perfringens (AK 1-9 Isolate).
Graphs were plotted from data produced by gas analyzer for O 2 , CO, H 2 , CO 2 and H 2 S. They illustrate three possible outputs of instrument from left to right: Rate of gas changes, gas accumulation and immediate gas concentration in% Parameters in the linear scale, O 2 values omitted.
The maximum amount of produced H 2 upon utilizing molasses as carbon source was observed by Clostridium perfringens (AK1-9 isolate) (4615µmol/l) Figure 6A, Table  1). Other isolates produced up to 100 times lower concentrations of H 2 ( Figure 6A, Table 1). This suggests that molasses is less universal substrate for H 2 production. The maximum amount of accumulated CO using molasses as carbon source was detected in Clostridium botulinum-like strain (AK 1-4 isolate). In Clostridium perfringens (AK 1-9 isolate) the CO gas evolution and/or consumption was inverse to that of H 2 ( Figure 6A, B). Also, smaller amount of CO but higher than of H 2 was detected in Clostridium difficile (AK 1-5 isolate).
Molasses as a waste product of sugar production was expected to be a good carbon source for bacteria. However, disappointing that bacteria did not grow well on molasses, with poor evolution of CO 2 (Figure 1-5). H 2 was evolved only with one isolate (AK 1-9). The reason of this failure did not analyze. Probably, high concentrations of Ca 2+ and the presence of many other carbon compounds besides sucrose disqualified molasses as good substrate for bacteria. Also the absence of ß-fructosidase activity in other isolates of bacteria could be the reason for failing to grow on the molasses. Accumulated H 2 production read out from Micro-Oxymax  Figure 5A were re-calculated for 1 l medium.

Conclusion
Five isolates were used to study the production of metabolic gases. Four isolates were from activated sludge and all belonged to the genus Clostridium: C. perfringens (AK 1-9 isolate), C. difficile (AK 1-5 isolate); one strain could be assigned to Clostridium spp. (AK 1-12 isolate); and one strain similar to C. botulinum (AK 1-4 isolate). The fifth was a rumen bacterium, which was identified as Escherichia coli (No 2-24 isolate). These isolates were cultivated anaerobically with molasses as complex carbon sources. Metabolic gases (O 2 , CO 2 , CO, H 2 , H 2 S and CH 4 ) were measured simultaneously by means of Micro-Oxymax gas analyzer with optical detection. It was found that all isolates produce CO 2 , CO, H 2 , and H 2 S dependent on the carbon source. Molasses was utilized only by AK 1-9 isolate.
Results suggest that the ability of tested bacteria to produce H 2 is universal. However, quantity of produced H 2 strongly depends on the carbon source. Comparative study of five isolated suggested that several aspects of utilization of carbon sources is highly variable between individual isolates. Thus, successful strain for the biotechnological use should be selected or modified in order to achieve optimal properties for the utilization of complex systems.