Fermentative Bio-Hydrogen Production of Food Waste in the Presence of Different Concentrations of Salt (Na+) and Nitrogen

Puleip Lee; Yuhoon Hwang  and Taejin Lee

doi:10.4014/jmb.1808.08023

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Research article

Environmental Microbiology and Biotechnology (EMB) | Environmental Microbiology

Fermentative Bio-Hydrogen Production of Food Waste in the Presence of Different Concentrations of Salt (Na⁺) and Nitrogen

Puleip Lee , Yuhoon Hwang and Taejin Lee ^*

Department of Environmental Engineering, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea

Correspondence to:

Taejin Lee
leetj@seoultech.ac.kr

Received: August 16, 2018; Accepted: November 30, 2018

J. Microbiol. Biotechnol. 2019; 29(2): 283-291

Published February 28, 2019 https://doi.org/10.4014/jmb.1808.08023

Abstract

Fermentation of food waste in the presence of different concentrations of salt (Na⁺) and ammonia was conducted to investigate the interrelation of Na⁺ and ammonia content in biohydrogen production. Analysis of the experimental results showed that peak hydrogen production differed according to the ammonia and Na+ concentration. The peak hydrogen production levels achieved were (97.60, 91.94, and 49.31) ml/g COD at (291.41, 768.75, and 1,037.89) mg-N/L of ammonia and (600, 1,000, and 4,000) mg-Na⁺/L of salt concentration, respectively. At peak hydrogen production, the ammonia concentration increased along with increasing salt concentration in the medium. This means that for peak hydrogen production, the C/N ratio decreased with increasing salt content in the medium. The butyrate/acetate (B/A) ratio was higher in proportion to the bio-hydrogen production (r-square: 0.71, p-value: 0.0006). Different concentrations of Na+ and ammonia in the medium also produced diverse microbial communities. Klebsiella sp., Enterobacter sp., and Clostridium sp. were predominant with high bio-hydrogen production, while Lactococcus sp. was found with low bio-hydrogen production.

Keywords

Ammonia, salt, bio-hydrogen, dark fermentation

Introduction

Hydrogen production technologies have gained significant attention due to the increased demand for hydrogen as an energy carrier [1]. The only by-product of combustion produced is hydrogen, making it a pollutant-free, alternative form of energy. Hydrolysis of chemical hydrides or metals has recently received attention as an optimal method for the production of hydrogen [2-4]. However, other chemical hydrogen production methods, such as steam reforming of hydrocarbons and partial oxidation of combustible fossil fuels, are energy intensive and require high temperature, resulting in the release of carbon dioxide and other pollutants as by-products [5]. Under these circumstances, biological hydrogen production from biomass appears to be one of the promising ways to replace traditional methods, as it can produce hydrogen competitively under ambient conditions, without causing pollution problems [6, 7].

Hydrogen can be generated through various biological production processes including direct bio-photolysis, indirect bio-photolysis, photofermentation, or dark fermentation [8]. Compared to the other three processes, the dark fermentation process has a high production rate, and much simpler operational requirements [9, 10]. Dark fermentation does not need light and has a very high evolution rate of hydrogen compared to photosynthetic processes [9]. Valuable by-products and biomass produced in the hydrogen fermentation make dark fermentation more favorable for commercialization [11, 12].

Even though dark fermentation of organic waste has high potential to produce bio-hydrogen together with the reduction of waste [13], low bio-hydrogen yield is a bottleneck to its practical application. Further research is needed on optimizing microbial community enrichment or the operational parameters of the reactor, such as pH, salt content, temperature, or H₂ partial pressure to improve bio-hydrogen yield [14]. Na⁺ is one of the parameters that affect bio-hydrogen production. It is originally a micronutrient for bacterial growth [15], but it is also related to hydrogen formation based on the Na⁺ gradient in cells. The Na⁺ gradient produced by high extracellular Na⁺ concentration enhances the reduction of oxidized ferredoxin, resulting in a higher concentration of reduced ferredoxin, which favors the formation of H₂ [16, 17]. Nevertheless, high Na⁺ concentrations could cause an inhibitory effect on biological hydrogen production because of changes in the metabolic pathway to favor the formation of other metabolites, rather than hydrogen production. [18-19]. Nitrogen is another affecting factor for hydrogen producers, as it is an important component of living matter [20]. Although ammonia nitrogen (from NH₃ or NH₄ ⁺) is an essential nutrient for bacterial growth, it has been reported to be toxic in dark fermentation processes if the concentration is higher than threshold [21, 22]. When NH₃ penetrates the cell membrane, it is protonized to NH₄ ⁺, which destroys the balance in intracellular pH [23]. But to date, little information is available about variations of bio-hydrogen production caused by the interrelation effect of two or more parameters in the sources.

Food and food processing wastes could be potential feedstock for biological hydrogen production because of the high content of organic matter and nutrients [24]. Food waste contains large portions of carbohydrate, protein, and fat, which can readily be degraded via dark fermentation. However, the composition of food waste is varied depending on its source. Most food waste may be lacking in nitrogen, but some food waste contains high amounts of salt and nitrogen [17, 25]. Therefore, in order to utilize food waste appropriately as a feedstock for dark fermentation, the effects of salinity or nitrogen content on the activities and metabolic processes of hydrogen production microbes need to be studied.

This study investigated the interrelation of the salt (Na⁺) and ammonia content on the fermentative bio-hydrogen production from food waste. For this purpose, the bio- hydrogen production efficiency of food waste with different Na and ammonia regimes was analyzed by a modified Gompertz equation. The formation of Volatile Fatty Acids (VFAs) under different conditions of salt (Na⁺) and ammonia content were also investigated. In addition, analysis of the microbial community involved in the fermentative bio-hydrogen production from food waste was conducted by denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified V3 region of 16S rDNA.

Materials and Methods

Microorganisms and Culture

The return activated sludge from a wastewater treatment plant was used as seed sludge for this study. The synthetic medium [26] contained 15 g/l of sucrose, 10 mg/l of CaCl₂•2H₂O, 100 mg/l of MgSO₄•7H₂O, 10 mg/l of NaCl, 2 g/l of NH₄HCO₃, 1 g/l of KH₂PO₄, 15 mg/l of MnSO₄•7H₂O, 10 mg/l of NaMoO₄•2H₂O, and 2.78 mg/l of FeCl₂. The 10 ml of seed sludge was added into 90 ml of the synthetic medium under anaerobic conditions [27]. The initial pH of the medium in serum bottles was set at 7.0. For the acclimation of seed microflora, the cultures were sequentially transferred from the serum bottles into fresh medium.

Operation of Batch Reactor

The substrate, or food waste, was obtained from a university cafeteria and was crushed by an electrical blender in an anaerobic glove box filled with nitrogen gas. Table 1 shows the characteristics of the food waste, from which all of the substrates were filtered through a stainless steel sieve (US Mesh No. 10). NH₄Cl and NaCl were analytical grade (Ducksan Co., Korea) and used as the nitrogen and Na source, respectively. Ammonia and Na⁺ concentrations of the medium were prepared in the range of (100– 5,000) mg-N/L of ammonia and (600–4,000) mg-Na⁺/L. Initial COD concentration was adjusted to be 10,000 mg/l. The carbon to nitrogen (C/N) ratio in the feed stock, which ranged (0.75–37.5), was artificially adjusted according to the purpose of the experiment.

Table 1 . Characteristics of collected food waste.

Parameter	Concentration (mg/L)
Total COD	321,788 ± 80,448
Soluble COD	201,117 ± 62,570
Total Nitrogen(T-N)	6,491 ± 292
Total Phosphorus(T-P)	397 ± 9
Total Carbohydrate	263,300 ± 1,571
Protein	9,447 ± 1,158
Na⁺	19,200 ± 546

Biological hydrogen production potential (BHP) tests of food waste were conducted in the batch reactor with a 1.5-L working volume. Fig. 1 is a schematic diagram of the batch reactor, which was stirred at 250 rpm using a magnetic stirrer. The headspace was initially maintained in an anaerobic condition using nitrogen gas. After that, 15 ml of seed solution was added, resulting in an initial volatile suspended solids (VSS) concentration of approxi- mately 10 mg/l. The reactor was kept at 30°C in the dark to avoid the growth of photosynthetic bacteria. The pH of the reactor was kept constant at 5.5 by the addition of KOH solution [27]. The generated biogas was collected by a gas collector on the reactor. At regular intervals, 0.2 ml of headspace gas was withdrawn for analysis.

Fig. 1. A sketch of the batch reactor for bio-hydrogen production.

Analytical Methods

The hydrogen and CO₂ concentrations were analyzed using a gas chromatography (GOW-MAC, USA), equipped with a thermal conductivity detector (TCD). The applied separation column and carrier gas were a 6’×1/8’’ stainless steel SS 350A Molsieve 13X and nitrogen gas at a flow rate of 30 ml/min, respectively. The injection port, column, and detector temperature were 80, 100, and 100°C, respectively.

The VSS for cell concentration, total or soluble chemical oxygen demand (COD), total nitrogen (T-N), and total phosphorus (T-P) were analyzed according to the standard methods [28, 29]. Carbohydrate and protein concentrations were measured by the phenol-sulfuric acid and modified Lowry methods, respectively [29, 30]. High-performance liquid chromatography (KNAUER, Germany) and a UV detector operating at 210 nm were used for VFAs (butyrate, acetate, lactate, and propionate) analysis. The column was a Supelcogel C-610H (130 mm × 7.8 mm ID). Ammonia and sodium concentrations were measured using ion chromatography (Metrohm compact 761, Switzerland).

Model Development

The cumulative bio-hydrogen production of the food waste was analyzed by the modified Gompertz equation (Eq. (1)) [27]

(1)

M = P \cdot \exp \{- \exp [\frac{R \cdot e}{P}] (λ - t) + 1\}

where, M and P are the cumulative hydrogen production (in ml) and the hydrogen production potential (ml), respectively. R is the maximum hydrogen production rate (ml/h), λ is the lag-phase time (h), e is exp(1)=2.718, and t is the incubation time (h).

Microbial Community Analysis

PCR was performed to assess the composition of the microbial community. DNA was extracted from microorganisms using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., USA). The eluted DNA from the microorganisms in the samples was reamplified by PCR with the forward primer 341f (5’-CCTACG GGAGGCAGCAG-3’) and the reverse primer 518r (5’-ATTACCGCGGCTGCTGG-3’) [31]. The quality of the DNA samples was analyzed by DGGE [32]. Sequencing analyses of the bands on the gradient gel were conducted for the closest reference microorganisms in the GenBank database using the BLAST program [33].

Results and Discussion

Bio-Hydrogen Production by Various Salt (Na⁺) Concentrations

A number of studies have reported the negative effect of Na⁺ and ammonia on hydrogen production with mixed cultures [17,34-38]. High Na⁺ concentrations exceeding the threshold level may result in the formation of other metabolites than hydrogen, which cause the suppression of bio-hydrogen production [19]. The low cell growth caused by this utilization of energy finally results in low hydrogen production [17]. High concentrations of ammonia have also been reported to be toxic on anaerobic digestion processes [37].

Several studies [16, 21, 38] reported that ammonia concentrations resulted in shifts to generate varying concentrations of soluble metabolites by altering metabolic pathways of the dark fermentation process. These end- products could also have a suppressive effect on bio- hydrogen production. Such a strategy as dilution of the reactor contents below suppressive threshold concentration, or microbial acclimation to the inhibitors prior to the fermentation process, could be proposed to reduce the inhibition of bio-hydrogen production. To optimize the strategies, it is worthwhile evaluating the interrelation of ammonia and Na⁺ concentrations on bio-hydrogen production, rather than evaluating the influence of independent factors.

The effects of Na⁺ and ammonia concentrations on bio-hydrogen production were investigated with medium containing in the range of (200–5,000) mg-N/L of ammonia and (600–4,000) mg-Na⁺/L. The hydrogen production curves over time were analyzed by Eq. (1). Table 2 lists the hydrogen production rates against the Na⁺ and ammonia concentrations.

Table 2 . Parameter values for bio-hydrogen gas with different salt (Na⁺) and ammonia concentrations in the food waste.

Sodium concentration (mg-Na⁺/L)	ammonia concentration (mg-N/L)	Production potential		H₂ yield (mL H₂/g COD)	Production rate		r²

		P_h(mL)	p-value		R_h (mL/g dry wt biomass/hr)	p-value
600	100	226.86	<0.0001	14.38	13.83	0.0017	0.98
	150	344.64	<0.0001	26.37	15.68	0.0009	0.98
	200	1268.67	<0.0001	102.00	83.43	<0.0001	0.99
	350	1416.06	<0.0001	93.33	94.29	0.0107	0.97
	600	848.27	<0.0001	59.64	18.14	<0.0001	0.98
	1500	379.89	<0.0001	19.64	21.83	0.0011	0.98
	3000	269.21	<0.0001	12.14	13.95	0.0265	0.96
	5000	203.88	<0.0001	13.41	25.52	0.0061	0.98

1000	200	775.20	<0.0001	51.97	36.37	0.0018	0.98
	600	1309.50	<0.0001	77.11	55.60	0.0769	0.92
	1500	579.57	<0.0001	37.44	41.11	<0.0001	0.99
	3000	291.53	<0.0001	16.86	20.74	0.0193	0.97

4000	100	514.28	<0.0001	41.85	19.12	<0.0001	0.99
	200	495.29	<0.0001	39.47	30.91	0.0767	0.98
	600	673.73	<0.0001	65.14	22.87	0.0148	0.97
	1500	661.55	<0.0001	40.45	27.19	0.0091	0.98
	3000	340.08	<0.0001	26.91	4.23	0.0007	0.96
	5000	241.90	<0.0001	18.09	14.50	0.0259	0.96

Fig. 2 plots the hydrogen production potential rate (P_h) against the ammonia concentration in the presence of Na⁺, and the relationship was obtained using a Weibull 5- parameter equation (r² = 0.97, Sigma Plot 2006 Systat Software Inc., USA). Analysis of the data showed that peak hydrogen production differed according to the ammonia and Na concentrations in the medium. The peak hydrogen production levels achieved were (97.60, 91.94, and 49.31) ml/g COD at (291.41, 768.75, and 1,037.89) mg-N/L of ammonia and (600, 1,000, and 4,000) mg-Na⁺/L of salt concentration, respectively. Since the initial COD value was 10,000 mg/l, C/N ratios at peak hydrogen production were 12.87, 4.88, and 3.61, respectively. This means that the presence of higher salt (Na⁺) content reduced the optimum C/N ratio for bio-hydrogen production. An earlier study [25] reported that peak hydrogen productivity reached a C/N-ratio of 47, but this experimental result implies that to maximize the production of bio-hydrogen, inhibition factors must be considered, because the C/N ratio could change according to the Na⁺ concentration, as shown above.

Fig. 2. Bio-hydrogen production profile in the various concentrations of ammonia and sodium (Na⁺). The initial concentration of COD at each mode was 10,000 mg/l. Symbols on the graph represent average values, of three experimental data sets. Error bar of experimental data was conducted, using Sigma Plot software.

Hydrogen production and substrate consumption could be inhibited in the presence of toxic substances while different degrees of inhibition would be shown, depending on the microbial species [15, 39, 40]. Our experimental results show increasing peak hydrogen production with lower sodium concentration. A previous study [41] reported that NADH-ferredoxin oxidoreductase activity could be enhanced in the presence of the proper concentration of Na⁺ for hydrogen production. This result suggests that bio- hydrogen production using food waste as a substrate could be more efficient when Na⁺ and ammonia concentrations are properly adjusted. It is also noteworthy that with high sodium (Na⁺) content in Fig. 2, the slope of cumulative hydrogen production against the ammonia concentration in the medium is much more gentle than at lower strength. We believe that no such study on bio-hydrogen generation that considers the complex correlation of nitrogen and salt (Na⁺) concentration has been published previously.

Variations of Individual VFAs Concentrations and B/A Ratio

Acid production is usually accompanied by hydrogen production, which in turn is coupled with the production of solvents, such as ethanol, methanol, butanol, propanol, and acetone. Thus, monitoring of these intermediates reflects the metabolic pathways of microorganisms. Table 3 shows VFA distributions and COD balance in the reactor. Carbon content in biomass was assumed at C5H7O2N. Others (%) in Table 3 mean the soluble COD minus the sum of VFAs and soluble carbohydrate concentration divided by the initial COD concentration, referring to solvent concentrations of products that were not detected by the UV detector by HPLC at 210 nm after reaction [27, 42]. Initial COD of 10,000 mg/l was converted to 100%. Values of other compounds are also converted into COD values, and divided by initial COD for recovery calculation. At various concentrations of Na⁺ and ammonia, acetate, butyrate, and propionate were the abundant species. As hydrogen production increased, concentrations of acetate and butyrate increased; in contrast, reduction of hydrogen production resulted in high propionate concentration.

Table 3 . Product distribution and recovery ratio on COD basis as a function of the Na⁺ and ammonia concentrations.

Sodium (mg-Na⁺/L)	Ammonia (mg-N/L)	Initial COD	Fatty acid					Biomass	Soluble COD	remain of carbohydrate	Others	H₂	Recovery	B/A ratio

			Acetate	Pro pionate	Formate	Butyrate	Lactate
600	150	100	0.32	N.D.	0.06	0.09	0.42	7.17	90.01	9.84	79.28	2.03	99.21	0.28
	200	100	1.11	N.D.	N.D	1.37	0.00	14.54	77.78	3.81	71.49	6.66	98.99	1.23
	350	100	0.88	N.D	N.D	2.53	1.57	16.02	75.76	6.63	64.16	8.53	100.30	2.88
	600	100	0.76	1.30	N.D	1.19	0.00	13.44	81.79	2.03	76.52	4.48	99.71	1.57

1000	200	100	3.09	1.93	0.34	1.66	N.D	15.43	88.01	2.54	78.45	4.45	107.89	0.54
	600	100	1.45	1.75	N.D	1.98	0.00	15.93	85.02	3.98	75.86	6.13	107.08	1.37
	1500	100	2.61	2.46	0.29	2.15	0.00	15.49	85.21	6.47	71.23	3.23	103.93	0.82
	3000	100	3.28	3.34	N.D	2.45	N.D	15.55	91.41	2.77	79.57	2.79	109.76	0.75

4000	100	100	2.34	1.17	0.79	1.21	0.00	19.24	81.50	9.21	66.78	3.48	104.22	0.52
	200	100	3.79	2.06	N.D	1.58	N.D	18.84	76.92	9.03	60.46	3.59	99.35	0.42
	600	100	4.10	2.11	0.00	2.90	0.00	17.48	84.04	9.91	66.60	4.60	106.11	0.71
	1500	100	3.56	2.61	N.D	3.80	2.08	14.31	84.97	5.47	67.44	3.45	102.73	1.07

Recovery = (soluble COD + H₂ + Biomass)/ initial COD×100 (%).

Fig. 3 shows the B/A ratio plotted and analyzed, with hydrogen production. The B/A ratio was higher in proportion to bio-hydrogen production (B/A ratio = 0.3242×Hydrogen production (%) - 0.4297, r-square: 0.71, p-value: 0.0006). Previous studies [27, 43] have reported that higher ratio of B/A, could be more preferable for hydrogen production. The B/A ratio change implied a microbial community shift, due to changes of ammonia or Na⁺ concentrations, resulting in lower production of hydrogen. This finding agreed with previous results, that in the presence of high Na⁺ concentration, hydrogen-producing bacteria displaced the fermentation process towards the acetate synthesis pathway, instead of the butyrate pathway [17].

Fig. 3. Changes of B/A ratio against the bio-hydrogen production. Hydrogen production (%) represents the produced bio-hydrogen gas from the initial substrate concentration (as COD). Regression of experimental data was conducted using Sigma Plot software. B/A ratio = 0.3242 × Hydrogen production (%) – 0.4297. (r-square: 0.71, and p-value: 0.0006 Straight line: regression fit, Dotted line: 95% confidence intervals)

PCR-DGGE Profile Analysis of the Microbial Community

The experimental results suggested that the microbial community changes depending on the Na⁺ and ammonia concentrations determines the changes in hydrogen production activities and acid production. The effect of Na⁺ and ammonia on the microbial community responsible for bio-hydrogen production was investigated and confirmed by PCR-DGGE analysis on 16s rDNA. The relative diversity of the microbial community and the degree of abundance of each microbial group are closely related with the number of bands and the band intensity in the gel, respectively [44]. Fig. 4 shows the PCR-DGGE profiles of each sample, which revealed band patterns in the presence of Na⁺ and ammonia.

Fig. 4. PCR-DGGE profile of the cultures at various concentrations of Na⁺ and ammonia. The numbers above the arrow on each lane represent the concentrations of Na⁺ and ammonia, and the B/A ratio, respectively.

Based on the band intensities, in 600 mg-Na⁺/L sodium concentration, bands (B, D, F, H, I, J, K, M, Q, S, and V) are present. In 1,000 mg-Na⁺/L sodium concentration, bands (A, B, C, E, N, P, Q, R, U, X, Y, Z, AA, and AB) are detected, indicating the existence of various microbial species. In 4,000 mg-Na⁺/L sodium concentration, bands (B and C) are clearly evident, while bands (G, L, N, O, T, W, and AA) are dimly visible. In 600 mg-Na⁺/L of sodium concentration, (F, H, I, J, K, M, Q, S, and V) are major bands with relatively high B/A ratio, representing high production of bio- hydrogen. In 1,000 mg-Na⁺/L, bands (C, P, U, X, and Y) are predominant, with high B/A ratio. In 4,000 mg-Na⁺/L, bands (N and R) are distinguishable with high B/A ratio. Taken together, under different Na⁺ concentrations, distinct band patterns with similar B/A ratios are evident. This reveals that each sample has microbial diversity according to the Na⁺ and ammonia profiles, even though it has a similar hydrogen production rate. Bands of interest were sequenced to investigate the microbial diversity among samples. The NCBI BLAST program was used to identify microbes and accession numbers from the 16S rDNA. Table 4 shows the closest affiliation and similarity of the bands on the gradient gel. Three major bacterial taxa were identified, with nine sequences related to the class γ-Proteobacteria, five affiliated with Bacteria, and fourteen related to the phylum Firmicutes.

Table 4 . The affiliation of DGGE bands as determined by the rDNA sequences.

Band name	Gen Band Search Result	Accession No.	Similarity	Taxonomic Description (Class)

	Phylogenetically Closet Relative
A	Enterococcus sp. HGB0104	JX519903.1	99	Firmicutes
B	Bacterium NLAE-zl-C199	JQ607927.1	95	Bacteria
C	Uncultured bacterium clone OC2_N15	KC860573.1	98	Bacteria
D	Lactococcus lactis isolate TIL8	JX893570.1	91	Firmicute
E	Clostridia bacterium S130(2)-2	GU136556.1	92	Firmicutes
F	Uncultured bacterium clone 70-1-2	DQ842542.1	100	Bacteria
G	Lactococcus lactis subsp. lactis strain NM151-4	HM218597.1	82	Firmicute
H	Klebsiella pneumonia strain ND6	JF927782.1	95	γ-proteobacteria
I	Clostridium saccharobutylicum DSM 13864	CP006721.1	90	Firmicute
J	Enterobacter hormaechei strain LAB 229	KJ156326.1	97	γ-proteobacteria
K	Enterobacter sp. NCCP-231	AB610883.1	94	γ-proteobacteria
L	Uncultured Enterobacteriaceae bacterium clone CSC13DXRNA 24	KJ624484.1	100	γ-proteobacteria
M	Clostridium algidicarnis strain E107	JX267120.1	86	Firmicute
N	Clostridium butyricum strain W5	DQ831124.1	100	Firmicute
O	Hydrogenoanaerobacterium sp. YN3	AB537982.1	98	Firmicute
P	Uncultured Clostridium sp. clone 8426	KF508692.1	90	Firmicutes
Q	Klebsiella sp. d252	FJ950657.1	86	γ-proteobacteria
R	Hydrogenoanaerobacterium sp. YN3	AB537982.1	98	Firmicutes
S	Uncultured bacterium clone B.11	KF512526.1	95	Bacteria
T	Klebsiella pneumonia strain FY2	KJ599860.1	94	γ-proteobacteria
U	Enterobacter sp. 2356	JX174233.1	98	γ-proteobacteria
V	Clostridium sp. R6	EF174500.1	91	Firmicute
W	Klebsiella sp. CCFM8383	KJ803940.1	95	γ-proteobacteria
X	Clostridium sp. MCF105	KC155326.1	98	Firmicutes
Y	Uncultured Clostridium sp. clone b2-73	JX575825.1	98	Firmicutes
Z	Enterobacter sp. 2358	JX174235.1	99	γ-proteobacteria
AA	Uncultured bacterium clone GDIC2IK01AKQ69	JF670002.1	85	Bacteria
AB	Uncultured Clostridium sp. clone T12	DQ168179.1	89	Firmicutes

Clostridia bacterium, Clostridium saccharobutylicum, Clostridium algidicarnis, Clostridium butyricum, and Clostridium sp. were detected as part of the Clostridium genus. Hydrogenoanaero- bacterium sp., Enterococcus sp., Lactococcus lactis, and Lactococcus lactis subsp. lactis were also identified. The Clostridium genus of the phylum Firmicutes showed comparatively high dominance, as shown in Fig. 4 by bands (E, I, M, N, P, V, X, Y, and AB), and had been reported to generate hydrogen gas under the fermentation process [33,45-48]. The genus in the class γ-proteobacteria were the Klebsiella and Enterobacter genus, which had also been known as hydrogen-producing bacteria [29]. The Klebsiella genus was shown in Fig. 4 in bands (H, Q, T, and W). The Enterobacter genus was also present in bands (J, K, L, U, and Z). They have been reported for hydrogen production [49–54]. Although Enterococcus sp. or Hydro-genoanaerobacterium sp. have been reported as hydrogen- producing microorganisms [55, 56], Lactococcus sp., designated as bands (D and G), is known as an inhibitor of hydrogen fermentation [57]. It is known that most of the microbes identified in this study are associated with hydrogen production, except for Lactococcus sp. This study conclusively shows that the interrelations of the ammonia and Na⁺ concentrations change the microbial community in the medium, and consequently show different hydrogen production rates and B/A ratios. The analysis of microbial communities with different concentrations of Na⁺ and ammonia shows various diversities for each condition. At 300 mg-N/L of ammonia and 600 mg-Na /L sodium concentration, Klebsiella sp., Clostridium sp., and Enterobacter sp. were predominant. At 800 mg-N/L of ammonia and 1,000 mg-Na /L sodium concentration, Clostridium sp., and Enterobacter sp. were the most abundant hydrogen- producing bacteria. At 1,500 mg-N/L of ammonia and 4,000 mg-Na⁺/L sodium concentration, Clostridium sp. and Hydrogenoanaerobacterium sp. were the microbes that were most responsible for hydrogen production.

Acknowledgments

This work is financially supported by the Korea Ministry of Environment (MOE) as a Knowledge-based environmental service waste to energy recycling human resource development project.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

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J. Microbiol. Biotechnol. 2019; 29(2): 283-291

Published online February 28, 2019 https://doi.org/10.4014/jmb.1808.08023