Article pubs.acs.org/EF
Mesophilic and Thermophilic Biohydrogen Production from Xylose at Various Initial pH and Substrate Concentrations with Microflora Community Analysis Chunsheng Qiu,*,†,‡ Yazhe Zheng,† Jianfeng Zheng,†,‡ Yan Liu,§ Chunyu Xie,† and Liping Sun†,‡ †
School of Environmental and Municipal Engineering, Tianjin Chengjian University, Tianjin 300384, PR China Tianjin Key Laboratory of Aqueous Science and Technology, Tianjin 300384, PR China § School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China ‡
ABSTRACT: Anaerobic dark fermentation biohydrogen production from xylose was investigated under mesophilic (35 °C) and thermophilic (55 °C) conditions at various initial pH (5.0−10.0) and substrate concentrations (2.5−12.5 g/L). In addition, the microbial community structure variations under different temperatures were analyzed. It was demonstrated that the maximum hydrogen yield (1.24 mol-H2/mol-xylose) was obtained with substrate concentration of 7.5 g/L and initial cultivation, pH 7.0, at 35 °C, with butyrate, acetate, and ethanol as the major byproducts. The increase of substrate concentration resulted in accumulation of volatile fatty acids (VFAs), especially propionate, and a decrease in final pH under mesophilic conditions. However, the hydrogen yield increased along with the increase of substrate concentration at 55 °C with butyrate and ethanol as the main metabolite. Stable pH of the system could be maintained even at high xylose concentration up to 12.5 g/L due to a low level of VFAs accumulation. A lower hydrogen yield of 1.14 mol-H2/mol-xylose was obtained at thermophilic condition, while a stable operation condition could be achieved and maintained more easily. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that microbial community structures of both systems were dominated with bacterial species related to Clostridium, while the thermophilic system had fewer hydrogen-producing microbial species than that at mesothermal condition.
1. INTRODUCTION In recent times, a great deal of attention is being paid to the usage of eco-friendly fuels from biomass that can reduce the negative environmental impacts by the use of fossil fuels. Lignocellulosic materials from agriculture and forest management are the largest sources of carbohydrates, mainly hexose and pentose, and possess the potential for biofuels production.1,2 Hexoses from lignocellulose can be effectively converted to various biofuels with relatively high yield and productivity, including bioethanol3 and biobutanol,4 as well as biogas.5,6 However, low yields and production rates have been major barriers to the bioconversion of pentose to liquid biofuels, due to the lack of effective microorganisms.7,8 Hydrogen is a promising energy carrier for its high energy yield and environmental friendliness. Hydrogen production from pentose (mainly xylose) through anaerobic dark fermentation is recognized as a potential and environmental friendly process and can be an effective way to utilize this substrate which is wasted otherwise.9−11 Xylose, one of the pentoses, is abundant in various kinds of lignocellulosic materials and wastes/wastewaters as a major component of hemicellulose and could serve as substrate for anaerobic dark fermentation.12,13 Research for biological production of hydrogen could be classified as pure culture and mixed culture fermentation. Xylose anaerobic fermentation by mixed culture is more efficient and practical for its remarkable advantages, including no requirements for pretreatment of the feedstock and the possibility of mixed substrates cofermentation for its high microbial diversity.13 Temperature © XXXX American Chemical Society
and pH are the two major environmental and operating factors in biological processes using mixed cultures.14−17 Hydrogen fermentation processes of xylose are readily affected by temperature changes because metabolic pathway and microbial community structure would be altered along with the temperature change. Different metabolic byproducts and hydrogen-producing species have been observed under mesophilic, thermophilic, and extreme thermophilic conditions in a biohydrogen production system.14,17,18 Meanwhile, pH could also affect process efficiency in hydrogen production activity, especially through liquid byproducts (organic acids and alcohols) distribution and concentration.16,19 In batch culture, the pH variation of the hydrogen fermentation process was usually caused by the accumulation and distribution of volatile fatty acids (VFAs) which were closely associated with the initial xylose concentration.16 Moreover, investigators have reported that byproducts could have an inhibitory effect on bacteria growth rate and hydrogen production.20 Thus, understanding the influence of temperature, substrate, and byproducts concentration, as well as the microbial community structure and pH variation, is necessary for xylose-related hydrogen fermentation applications. Few comprehensive studies on xylose based hydrogen production using mixed cultures have been reported. Received: September 20, 2015 Revised: January 7, 2016
A
DOI: 10.1021/acs.energyfuels.5b02143 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels
amplicon was estimated on 1.5% agarose gel. Primers 338f and 518r with 40 bp GC clamp at the 5′ end (5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTA CGGGAGGCAGCAG-3′/5′-ATTACCGCGGCTGCTGG-3′) were used to amplify the fragment of the V3 region in a second PCR, under conditions of initial denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 90 s, with final extension at 72 °C for 5 min. PCR products were stored at 4 °C and analyzed on 1.5% agarose before DGGE. DGGE analysis of the amplicons obtained from the second PCR was performed using the DGGE system (D-Code, Bio-Rad) with 8% (v/v) polyacrylamide gels and a denaturant gradient of 30−60%.24 Electrophoresis was performed for 7 h at 150 V in a 0.5× TAE buffer at 60 °C. DGGE gels were stained with 0.1% AgNO3 solution for 25 min and analyzed on a Universal Hood II system (Bio-Rad). Most of the bands were excised from the gel and reamplified with primers 338f and 518r without a GC clamp. After reamplification, PCR products were purified with a TIANprep Midi Purification Kit (TIANGEN, China) and sent to a genetic analysis service company (BGI, China) for analyzing DNA sequences. The closest matches for partial 16S rRNA gene sequences were identified by database searches in Gene Bank using BLAST.18 2.4. Analyses. VSS was analyzed in accordance with the standard methods.25 Xylose was analyzed by the phenol-sulfuric acid method.26 The evolved biogas from vials was collected in sealed plastic bags. Its volume was measured by syringe (every 4 h) with calibrations and was calibrated to the volume at standard temperature of 0 °C and pressure of 1 atm. The biogas components (H2, CH4, and CO2) were measured by gas chromatography (GC) (Clarus 5800, PerkinElmer US) equipped with a thermal conductivity detector.27 The liquid samples were taken from the vials; the medium after culture was centrifuged at 12 000 rpm for 10 min, and the supernatant was used for the following soluble byproducts analyses. Samples were first acidized by phosphoric acid; the VFAs and alcohol were measured using a GC (Clarus 5800, PerkinElmer US) equipped with a flame ionization detector.28 The hydrogen yields were calculated on the basis of the degraded xylose. C balance of xylose degradation products was made on the basis of the measured data (average of triplicates).
On the basis of these considerations, this research was aimed at investigating the hydrogen-production using anaerobic sewage sludge microflora at various initial pH and xylose concentrations at mesophilic (35 °C) and thermophilic (55 °C) conditions (considering the subsequent methane fermentation). The pH values, temperature, and substrate concentration effects on the hydrogen yield, gas content, metabolite distribution, and so on were investigated in batch experiments. Furthermore, the community structures variation of hydrogen producing microflora were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE).
2. MATERIALS AND METHODS 2.1. Seed Microflora and Substrate. The seed sludge was the mixture of thickened sludge collected from a municipal sewage treatment plant and digested cow manure from a local farm (Tianjin, PR China) for various hydrogen producing bacteria contained in it and then screened with a sieve (diameter, 2.35 mm) to eliminate large particulate materials. The volatile suspended solid (VSS) of the screened sludge was 46.3 g/L. Before being seeded into the bioreactor, the screened sludge was heat-treated at 100 °C for 45 min to inhibit methane-producing bacteria. Xylose solution was used as carbon source, at concentrations of 10.0, 20.0, 30.0, 40.0, and 50.0 g/L, respectively. Sufficient inorganics were prepared in the substrate to provide the essential nutrients and trace elements for H2 producing consortia: 382.1 mg/L NH4Cl, 87.7 mg/L KH2PO4, 260.0 mg/L CaCl2·2H2O, 320.0 mg/L MgSO4·7H2O, 125.0 mg/L FeSO4·7H2O, 0.3397 mg/L Zn2+, 0.3365 mg/L Ni2+, 1.5747 mg/L Co2+, 0.2661 mg/L B3+, 2.8646 mg/L Mn2+, 5.7380 mg/ L I−, 0.1920 mg/L Cu2+, and 0.5950 mg-L Mo6+.11,21,22 2.2. Experimental Procedures. 250 mL serum vials were used for hydrogen production experiments. The vials were seeded with 150 mL of heat-pretreated sludge and fed with 50 mL of substrate of different xylose concentrations (10.0−50.0 g/L), equivalent to the final concentrations of 2.5, 5.0, 7.5, 10.0, and 12.5 g/L in the mixed culture. For the initial culture pH determination, a preliminary experiment was carried out at a xylose concentration of 7.5 g/L at 35 and 55 °C. The tested pH values were 5.0−10.0 in an interval of 1 unit. To investigate the effect of substrate concentration, pH of the solution was adjusted to 7.0 by HCl or NaOH (2 mol/L). Then, the vials were sealed with rubber stoppers and aluminum crimps. Pure nitrogen gas was purged into the sealed vials with needles inserted through the stoppers for 5 min to provide anaerobic conditions. The sealed vials were placed in water bath shakers fixed at 150 rpm. The temperature of the shakers was preset at 35 and 55 °C, respectively. The water bath shakers were stopped until no more hydrogen was detected; the supernatants were removed after 24 h of sludge settling, and 50 mL of fresh substrate was added. Distilled water was also added to the vials to maintain the total volume of 200 mL. Repeated transfer was stopped when no hydrogen yield increase compared to the previous cultivation was observed. Assays with seed sludge alone were used as blank controls. The evolved biogas was collected by polyethylene bags. Biogas produced from inoculum was subtracted from the sample assays. The experiment was conducted in triplicate. 2.3. Microbial Community Structure Analysis. To confirm hydrogen-producing bacteria species, PCR-DGGE was used to study microbial community structure in the mixed culture under different temperatures. The hydrogen-producing sludge samples were taken from the mixed culture with highest hydrogen yield at both temperatures during the repeated enrichment culture stage. The sample of the heat-treated seed sludge was also taken for PCR-DGGE analysis. Total DNA of the sludge samples was extracted with a Soil DNA Isolation kit (Soil gDNA kit, Biomiga, USA). PCR of eubacteria was then performed with the primers 27F and 1492R,23 under conditions of initial denaturation at 94 °C for 4 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 90 s, with final extension at 72 °C for 5 min. The reactions were subsequently cooled to 4 °C. The size of the
3. RESULTS AND DISCUSSION 3.1. Effects of Initial Cultivation pH on Hydrogen Production. Figure 1 shows the variation of hydrogen production and xylose consumption at various initial cultivation pH values. It is known that at pH 5.0 and 10.0 low substrate degradation rate and hydrogen production were found at 35 °C. While at 55 °C, this phenomenon was only found at an initial cultivation pH of 5.0. For other pH values’ cultivation, relatively high hydrogen production was obtained with almost complete degradation of xylose. At a pH of 7.0, peak hydrogen volumes of 294.6 and 344.6 mL were obtained at both temperatures, respectively. Since the other characteristics of the mixed culture, including initial oxidation−reduction potential, alkalinity, medium components, and so on, were in the ranges favoring hydrogen production, the differences in hydrogen production can be considered as the result of varied initial pH. The optimal initial pH (7.0) differed from some previous reports on hydrogen production from xylose or other carbohydrates.2,15,16,19 In the hydrogen fermentation process, the responsible microorganisms are mainly Clostridium species, which are able to grow in a pH range of 4.5−7.0.20 Despite the variation of initial pH in this study, the final pH values of most mixed cultures (4.5−7.0) were in the growth ranges of hydrogen-producing bacteria. For mixed culture fermentation, the presence of hydrogen-consuming organisms would greatly affect the hydrogen yield.20 The differences of the optimal initial pH reported may result from the mixed culture possibly B
DOI: 10.1021/acs.energyfuels.5b02143 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels
Figure 2. Hydrogen production performance of various initial xylose concentrations at 35 °C. (The xylose concentration and hydrogen content were analyzed after 84 h of fermentation when no more hydrogen was detected in all of the vials.)
variation of byproducts during the xylose bioconversion process as shown in Table 1. Liquid product analysis showed that the VFAs and ethanol were the main soluble microbial products. It can be seen in Table 1 that, at xylose concentration of 2.5−7.5 g/L, butyrate was the main metabolic intermediate byproduct followed by ethanol and acetate, while at 10.0 and 12.5 g-xylose/L, propionate became the major microbial product (up to 1578.3 mg/L) except for butyrate. It can also be seen that the hydrogen production variation trend was identical to ethanol production, which indicated that ethanol-producing microorganisms might be one species of the dominant bacteria. Along with the increase of xylose concentration, the final pH of the mixed culture decreased from 6.33 to 4.47 due to the increase of VFAs concentration, especially the accumulation of propionate which resulted in no hydrogen production. The increase of propionate concentration indicated a metabolic pathway shift between mixed acids and ethanol type fermentation. This phenomenon suggests that hydrogen production through ethanol fermentation was partially inhibited by high loading rate of the substrate and decrease of the pH. The hydrogen inhibition could also have resulted from a shift in the metabolic pathway during the decrease of pH in the liquid phase. It has been reported that bioactivity and metabolic pathway of hydrogen-producing species was affected at low pH (below 5.5).16 Meanwhile, high initial substrate concentration has led to accumulation of organic byproducts which has probably resulted in an unfavorable thermodynamic state that prevented further substrate degradation.30 A similar phenomenon has been reported using xylose at a concentration of COD 20 g/L when initial cultivation pH is over 7.0.14 Relatively high pH would be beneficial to hydrogen production through the ethanol fermentation process. 3.3. Effect of Substrate Concentration on Hydrogen Production at 55 °C. As for thermophilic biohydrogen production process (Figure 3), high xylose degradation rate (over 95.1%) was achieved at each tested xylose concentration. Compared with the results of the mesophilic condition, much higher hydrogen yield of 1.11 and 1.14 mol/mol-xylose was achieved at the same xylose concentration of 10.0 and 12.5 g/L, respectively. It suggests that hydrogen fermentation was not inhibited by high loading rate of the substrate, probably because the release of H2/CO2 to the headspace is more favorable at
Figure 1. Hydrogen production performance at various initial cultivation pH values. (The xylose concentration and hydrogen content were analyzed after 120 h of fermentation when no more hydrogen was detected in all of the vials.)
containing some hydrogen-consuming organisms from the different inoculum sources. GC analysis showed that the major liquid products were butyrate (267.8−2144.8 mg/L), acetate (278.9−644.8), and ethanol (40.0−256.6 mg/L) at 35 °C. While at 55 °C, an obvious increase of ethanol (277.1−994.6) and acetate (701.7−1298.1 mg/L) concentrations was found, with a decrease of butyrate (352.6−1586.1 mg/L) and other VFAs (data not shown) concentrations. Correspondingly, this resulted in a significant fluctuation of the final pH (4.2−8.1) at 35 °C and a stable final pH (4.5−5.4) at 55 °C. According to the data, we concluded that 7.0 was the optimal initial cultivation pH with hydrogen yields of 0.96 and 1.03 mol/molxylose at 35 and 55 °C, respectively. 3.2. Effect of Substrate Concentration on Hydrogen Production at 35 °C. Hydrogen production and metabolite distribution were investigated in the batch cultivations at different initial xylose concentrations of 2.5−12.5 g/L. As shown in Figure 2, a nearly complete degradation of xylose (over 95.6%) was observed at the initial xylose concentration between 2.5 and 7.5 g/L, and the highest hydrogen yield of 1.24 mol/mol-xylose occurred at 7.5 g/L substrate concentration with the max cumulative hydrogen volume (265.42 mL) and xylose degradation rate (96.24%) simultaneously. Hydrogen fermentation was inhibited with higher initial xylose concentration, which was probably due to the increase of hydrogen partial pressure.29 However, at initial xylose concentration of 10.0 and 12.5 g/L, the substrate was not completely removed (72.7 and 66.4% degradation rate, respectively), with lower hydrogen yield. This might be caused by the sharp decrease in final pH of the mixed culture and the C
DOI: 10.1021/acs.energyfuels.5b02143 Energy Fuels XXXX, XXX, XXX−XXX
Article
8.5 3.5 19.5 8.9 9.6 112.5 1.0 ± ± ± ± ± ± ± 290.5 47.7 952.6 565.8 256.6 2025.1 22.3 94.9 13.2 2.1 21.6 19.2 15.4 45.5 0.9 ± ± ± ± ± ± ± 110.9 47.7 570.6 865.0 151.4 1125.9 23.4 86.2 5.6 3.2 9.2 14.3 9.5 90.3 1.1 ± ± ± ± ± ± ± 84.8 47.4 336.5 452.3 223.6 1658.4 21.5 84.3 70.5 26.2 125.6 315.2 85.4 625.4 27.5 82.7
2.2 3.2 7.8 11.7 4.6 28.5 0.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.7 3.1 5.9 4.5 3.3 33.3 0.6 81.0 32.5 135.8 128.4 52.1 805.2 20.8 88.3
initial pH final pH consumed xylose (mg/L) CO2 (mL) H2 content (%) ethanol (mg/L) acetate (mg/L) propionate (mg/L) butyrate (mg/L) pentanoate (mg/L) C recovery (%)b
C recovery was calculated on the basis of the average value. bC recovery percentage (%) = total carbon content of all the byproducts (TC)/carbon content of the consumed xylose (XC) × 100%. XC (mg/L) = [(12.01 × 5)/150.13] × consumed xylose (mg/L).TC (mg/L) = [(12.01 × 2)/46.07] × ethanol (mg/L) + [(12.01 × 2)/60.05] × acetate (mg/L) + [(12.01 × 3)/74.00] × propionate (mg/L) + [(12.01 × 4)/88.11] × butyrate (mg/L) + [(12.01 × 5)/102.13] × pentanoate (mg/L) + (12.01 × 2) × [CO2 (mL) × 5/22.41].
10.1 0.9 13.2 16.3 55.2 59.4 0.5 173.9 49.4 798.2 701.2 1125.8 2025.4 20.7 97.2 9.5 2.3 15.6 67.7 7.6 67.9 0.3 ± ± ± ± ± ± ± 204.7 42.9 922.2 1520.3 161.6 1624.0 13.7 90.4
7.03 ± 0.05 6.63 ± 0.03 7214.8 ± 156.2 7.03 ± 0.01 5.81 ± 0.04 7217.9 ± 120.5 7.01 ± 0.05 6.70 ± 0.03 4760.1 ± 116.2 7.02 ± 0.03 5.95 ± 0.01 4769.4 ± 85.7 7.05 ± 0.02 6.73 ± 0.03 2353.5 ± 36.2 7.02 ± 0.02 6.33 ± 0.02 2313.4 ± 20.5
2.5
Figure 3. Hydrogen production performance of various initial xylose concentrations at 55 °C. (The xylose concentration and hydrogen content were analyzed after 84 h of fermentation when no more hydrogen was detected in all of the vials.)
higher temperature and the stable pH maintained in the mixed culture.29 Furthermore, ethanol production by the thermophiles showed a coincident tendency with the hydrogen production (Table 1). Lin et al. has also found that high fermentation temperature (55 °C) caused the buildup of ethanol rather than VFAs.14 Ethanol type fermentation has been proven to have high hydrogen yield and less VFAs accumulation.31−33 It can also be seen that the concentrations of propionate increased slightly along with the increase of substrate concentration, which resulted in the relatively stable pH of the mixed culture. As pH had a significant effect on the byproducts distribution and was important for the further design of the hydrogenproducing process, it is obvious that the thermophilic condition would have more advantages for maintaining a stable fermentation environment. Meanwhile, compared with the process at 35 °C, propionate (byproduct of the hydrogen consumed pathway) and pentanoate maintained low concentrations, which resulted in the stable hydrogen yield at initial xylose concentrations up to 10.0 and 12.5 g/L. In addition, relatively low H2 content detected at a xylose concentration of 2.5 g/L at both temperatures, as well as the C recovery percentage, might be due to the consumption of substrate through cellular respiration and the microbes’ growth. Figure 4 depicts the time course of hydrogen production during 72 h of fermentation at both cultivation temperatures. Despite a little longer lag phase, the thermophilic fermentation had a much higher hydrogen production rate and xylose loading rate than that of mesophilic fermentation. Compared with the whole fermentation period of 56 h at 35 °C, hydrogen production could be finished in 40 h at 55 °C. It has been previously reported that thermophilic fermentation of 50−55 °C had a higher hydrogen production rate.14 This could be attributed to higher bioactivity in the thermophilic range. The hydrogen yield value was comparable to that of mixed culture enriched from activated sludge, with hydrogen yield of 0.4−1.4 mol-H2/mol-xylose at mesophilic and thermophilic temperatures. Acetate, butyrate, and ethanol were also found to be the main liquid byproducts in these studies. 14,17 Furthermore, the hydrogen production efficiency was lower than that of hydrogen production using a pure culture T. neapolitana DSM 4359 at 75 °C(1.8−2.8 mol-H2/molxylose).34 The difference could be contributed to the culture
a
25.3 1.9 88.6 82.6 9.8 112.1 0.4 ± ± ± ± ± ± ± 457.2 47.7 1956.9 2011.2 255.6 2856.9 22.9 96.7 344.7 48.5 1658.8 1563.2 215.5 2165.3 18.3 94.9
21.3 1.8 73.4 75.1 9.4 87.5 0.5
217.4 39.5 552.3 756.4 1578.3 2135.8 21.4 93.9
15.5 2.3 22.2 24.0 57.5 66.4 0.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.01 ± 0.03 6.47 ± 0.02 12265.0 ± 88.2 7.02 ± 0.02 4.47 ± 0.01 8299.2 ± 90.5 7.00 ± 0.02 6.44 ± 0.02 9768.9 ± 136.8 7.05 ± 0.03 5.49 ± 0.02 7268.0 ± 90.5
12.5 35 °C 55 °C 10.0 35 °C 55 °C 7.5 35 °C 55 °C 5.0 35 °C 55 °C 35 °C
xylose (g/L)
Table 1. pH of the Mixed Culture, H2 Content, CO2 Production, and Liquid Metabolite Distributions of Various Initial Xylose Concentrations at 35 and 55 °Ca
55 °C
Energy & Fuels
D
DOI: 10.1021/acs.energyfuels.5b02143 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels
Figure 4. Daily variations in cumulative hydrogen production in batch culture at 35 and 55 °C (initial pH, 7.0; initial xylose concentration of 7.5 g/L at 35 °C and 12.5 g/L at 55 °C).
type (mixed vs pure cultures) and byproducts distribution. The enriched mixed culture used in our study included hydrogenconsuming organisms, because the increasing amounts of propionate were detected along with the increasing xylose concentration at both temperatures, which was the main product of hydrogen-consuming organisms.11 Meanwhile, acetate was the dominant byproduct in the pure culture fermentation system,34 and the theoretical maximum hydrogen yield of 3.3 mol-xylose could be achieved when only acetate is produced as liquid product.11 The method of inhibiting hydrogen-consuming organisms (besides methane producing bacteria) needed to be further improved. Moreover, although this mixed-culture fermentation system had lower hydrogen yield than the pure-culture system, it is attractive when wastes or wastewater containing various components are used as substrates.14 3.4. Bacterial Community Composition. To show the temperature effect on the microbial community composition, a DGGE analysis for the sludge samples at 35 and 55 °C, as well as the inoculum, was conducted. The DGGE profiles were presented in Figure 5, and the results of the sequence affiliation were shown in Table 2. As shown in Figure 5, the DGGE profiles of the microflora from the inoculum and slurries cultured at 35 and 55 °C were apparently different. A change in biohydrogen system temperature has not only altered the substrate utilization process efficiency, hydrogen production activity, and liquid product distribution, but it also caused the succession of microbial community. Meanwhile, the inoculum contained more bands suggesting that heat-treatment and repeated culture enriched certain microbial species associated with hydrogen production, thus reducing species diversity. Furthermore, the thermophilic culture showed its own characteristic profile with fewer bands compared with the inoculum and mesophilic culture. Twenty bands of the nucleotide sequences in the gel were successfully sequenced; Clostridium spp. was the dominant species at both 35 and 55 °C (Table 2). The affiliates of the genus Clostridium are identified as the major species for hydrogen production through dark fermentation and can be obtained by the heat treatment of biological sludge.35,36 A microorganism closely related to Clostridium thermopalmarium was present both at 35 and 55 °C (bands 6, 9, 10, 14, and 16). This strain could be isolated from palm wine with the optimum growth temperature at 55 °C; it was found to ferment sugars to butyric acid, acetic acid, hydrogen, and carbon
Figure 5. DGGE analyses of the microbial communities in the slurry. Lane 1 indicates the profile of the microflora of the inoculum; lanes 2 and 3 indicate the profiles of the microflora cultured at 35 and 55 °C, respectively.
dioxide.37 Clostridium saccharobutylicum (band 11) was found only at 35 °C. It was reported that hydrogenogenesis by dark fermentation in batch cultures of this strain was optimal at about 35 °C and an initial pH of 6.5, with butyrate and acetate as main byproducts.38 In addition, some subspecies of Clostridium saccharobutylicum could be applied for acetone− butanol−ethanol fermentation using sugar-based feedstocks, such as cane molasses and corn stover hydrolysate (containing xylose),39 which might also be one of the metabolic pathways for ethanol production. Band 20 (only found in thermophilic culture) showed 98% identity to unclassified Thermoanaerobacteriaceae bacterium. Several members of the genus Thermoanaerobacterium belonging to the family Thermoanaerobacteriaceae were reported to have the ability for hydrogen fermentation at an optimal temperature of 60 °C using xylose and glucose as substrate, with ethanol and acetate as the main byproducts.40 In addition, a thermophilic strain isolated from E
DOI: 10.1021/acs.energyfuels.5b02143 Energy Fuels XXXX, XXX, XXX−XXX
Energy & Fuels
■
Table 2. Characteristics of 16S rDNA Fragments Obtained from DGGE Gel band no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
closest relatives Bacteroidetes bacterium enrichment culture clone L35B_118 Clostridiales bacterium GluBS11 Clostridiales bacterium GluBS11 uncultured bacterium clone EPS003BBFL_86 uncultured bacterium clone D5_BP3 Clostridium thermopalmarium strain NMY5 uncultured Bacilli bacterium clone R1-61 uncultured bacterium MAAB_11.1.1 Clostridium thermopalmarium strain NMY5 Clostridium thermopalmarium strain NMY6 Clostridium saccharobutylicum DSM 13864 Clostridium sp. enrichment culture OTU196 Clostridium sp. YN5 Clostridium thermopalmarium strain HL-5 uncultured bacterium isolate DGGE band CES 11 Clostridium thermopalmarium strain KUM1 uncultured Prolixibacter sp. clone ADINDWW-B1 uncultured bacterium, isolate 181, clone CMW-181 uncultured bacterium isolate DGGE gel band HRB1 Thermoanaerobacteriaceae bacterium 91bM
similarity (%)
JF946993
99 98 99
KP233894 KP233894 JX521107
94 98
JN813764 EF639852
98 94 100
KP717536 KC785500 EF639852
96
EF639853
99
CP006721
98
KF812563
100 98 97
AB537983 KM036191 KP101334
99
HM756303
99
KM484759
98
FR775403
99
HQ231785
98
GU129121
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel.: +86 13612149816. Fax: +86 22 23085117.
accession no.
99
Article
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors wish to acknowledge the financial support provided by the National Natural Science Foundation of China (No. 21206106), the High School Science and Technology Fund Planning Project of Tianjin education committee, China (No. 20130515), and the research fund of Tianjin Science and Technology Committee (14ZCDGSF00032).
■ ■
ABBREVIATIONS GC = gas chromatography PCR-DGGE = polymerase chain reaction-denaturing gradient gel electrophoresis VFAs = volatile fatty acids VSS = volatile suspended solid REFERENCES
(1) Singh, N. B.; Kumar, A.; Rai, S. Potential production of bioenergy from biomass in an Indian perspective. Renewable Sustainable Energy Rev. 2014, 39, 65−78. (2) Nissila, M. E.; Lay, C. H.; Puhakka, J. A. Dark fermentative hydrogen production from lignocellulosic hydrolyzates-A review. Biomass Bioenergy 2014, 67, 145−159. (3) Gupta, A.; Verma, J. P. Sustainable bio-ethanol production from agro-residues: A review. Renewable Sustainable Energy Rev. 2015, 41, 550−567. (4) Green, E. M. Fermentative production of butanol-the industrial perspective. Curr. Opin. Biotechnol. 2011, 22 (3), 337−343. (5) Fang, F.; Mu, Y.; Sheng, G. P.; Yu, H. Q.; Li, Y. Y.; Kubota, K.; Harada, H. Kinetic analysis on gaseous and aqueous product formation by mixed anaerobic hydrogen-producing cultures. Int. J. Hydrogen Energy 2013, 38 (35), 15590−15597. (6) Jung, K. W.; Kim, D. H.; Shin, H. S. Application of a simple method to reduce the start-up period in a H2-producing UASB reactor using xylose. Int. J. Hydrogen Energy 2013, 38 (18), 7253−7258. (7) Kumari, R.; Pramanik, K. Improved bioethanol production using fusants of Saccharomyces cerevisiae and xylose-fermenting yeasts. Appl. Biochem. Biotechnol. 2012, 167 (4), 873−884. (8) Harner, N. K.; Wen, X.; Bajwa, P. K.; Austin, G. D.; Ho, C. Y.; Habash, M. B.; Trevors, J. T.; Lee, H. Genetic improvement of native xylose-fermenting yeasts for ethanol production. J. Ind. Microbiol. Biotechnol. 2015, 42 (1), 1−20. (9) Tyagi, V. K.; Lo, S. L. Microwave irradiation: A sustainable way for sludge treatment and resource recovery. Renewable Sustainable Energy Rev. 2013, 18, 288−305. (10) Cheng, X. Y.; Liu, C. Z. Hydrogen production via thermophilic fermentation of cornstalk by Clostridium thermocellum. Energy Fuels 2011, 25 (4), 1714−1720. (11) Wu, S. H.; Lin, C. Y.; Lee, K. S.; Hung, C. H.; Chang, J. S.; Lin, P. G.; Chang, F. Y. Dark fermentative hydrogen production from xylose in different bioreactors using sewage sludge microflora. Energy Fuels 2008, 22 (1), 113−119. (12) Ballesteros, I.; Negro, M. J.; Oliva, J. M.; Cabanas, A.; Manzanares, P.; Ballesteros, M. Ethanol production from steam-
hot springs, which also belongs to this family, was reported to be capable of degrading a wide range of carbon sources (including xylose) for ethanol production,41 which might be one of the possible reasons for the relatively high ethanol concentration detected at 55 °C.
4. CONCLUSIONS This study demonstrated that liquid byproducts distributions, pH of the mixed culture, and bacterial community composition were readily affected by the operating temperature and substrate concentrations in the anaerobic fermentative hydrogen production process from xylose. Mesophilic hydrogen fermentation of 35 °C had a relatively higher hydrogen yield at substrate concentration of 7.5 g/L, while thermophilic fermentation resulted in stable pH of the system even at high xylose concentration (12.5 g/L) with less VFAs accumulation. Butyrate, ethanol, and acetate were the major components of soluble metabolite products. At high substrate concentration, propionate was accumulated in the mesophilic fermentation process, while at 55 °C hydrogen production through ethanol type and butyrate type fermentation were not inhibited with relatively low propionate concentration detected. Although both systems were dominated with bacterial species related to Clostridium, the change of the biohydrogen fermentation temperature has caused the succession of the microbial community. Thermophilic fermentation was beneficial to the enrichment of hydrogen-producing bacteria and biodegradation of the substrate in high concentrations. F
DOI: 10.1021/acs.energyfuels.5b02143 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels explosion pretreated wheat straw. Appl. Biochem. Biotechnol. 2006, 130, 496−508. (13) Wang, W.; Xie, L.; Chen, J. R.; Luo, G.; Zhou, Q. Biohydrogen and methane production by co-digestion of cassava stillage and excess sludge under thermophilic condition. Bioresour. Technol. 2011, 102 (4), 3833−3839. (14) Lin, C. Y.; Wu, C. C.; Hung, C. H. Temperature effects on fermentative hydrogen production from xylose using mixed anaerobic cultures. Int. J. Hydrogen Energy 2008, 33 (1), 43−50. (15) Zhao, C. X.; Lu, W. J.; Wang, H. T. Simultaneous hydrogen and ethanol production from a mixture of glucose and xylose using extreme thermophiles I: Effect of substrate and pH. Int. Int. J. Hydrogen Energy 2013, 38 (23), 9131−9136. (16) Lin, C. Y.; Hung, C. H.; Chen, C. H.; Chung, W. T.; Cheng, L. H. Effects of initial cultivation pH on fermentative hydrogen production from xylose using natural mixed cultures. Process Biochem. 2006, 41 (6), 1383−1390. (17) Lin, C. Y.; Wu, C. C.; Wu, J. H.; Chang, F. Y. Effect of cultivation temperature on fermentative hydrogen production from xylose by a mixed culture. Biomass Bioenergy 2008, 32 (2), 1109−1115. (18) Kongjan, P.; O-Thong, S.; Kotay, M.; Min, B.; Angelidaki, I. Biohydrogen production from wheat straw hydrolysate by dark fermentation using extreme thermophilic mixed culture. Biotechnol. Bioeng. 2010, 105 (5), 899−908. (19) Khanal, S. K.; Chen, W. H.; Li, L.; Sung, S. W. Biological hydrogen production: effects of pH and intermediate products. Int. J. Hydrogen Energy 2004, 29 (11), 1123−1131. (20) Ciranna, A.; Ferrari, R.; Santala, V.; Karp, M. Inhibitory effects of substrate and soluble end products on biohydrogen production of the alkalithermophile Caloramator celer: Kinetic, metabolic and transcription analyses. Int. J. Hydrogen Energy 2014, 39 (12), 6391− 6401. (21) Demirel, B.; Scherer, P. Trace element requirements of agricultural biogas digesters during biological conversion of renewable biomass to methane. Biomass Bioenergy 2011, 35 (3), 992−998. (22) Yu, H. Q.; Fang, H. H. P.; Tay, J. H. Effects of Fe2+ on sludge granulation in upflow anaerobic sludge blanket reactors. Water Sci. Technol. 2000, 41 (12), 199−205. (23) Lane, D. J. 16S/23S rRNA sequencing; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley and Sons, Inc: New York, 1991; pp 115−175. (24) Zoetendal, E. G.; Akkermans, A. D. L.; Akkermans-van Vliet, W. M.; de Visser, J.; de Vos, W. M. The host genotype affects the bacterial community in the human gastrointestinal tract. Microb. Microb. Ecol. Health Dis. 2001, 13 (3), 129−134. (25) APHA. Standard methods for the examination of water and wastewater, 19th ed.; American Public Health Association: New York, 1995. (26) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350−356. (27) Roy, S.; Ghosh, S.; Das, D. Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry. Int. J. Hydrogen Energy 2012, 37 (21), 15867− 15874. (28) Playne, M. J. Determination of ethanol, volatile fatty acids, lactic and succinic acids in fermentation liquids by gas chromatography. J. Sci. Food Agric. 1985, 36, 638−644. (29) Angenent, L. T.; Karim, K.; Al-Dahhan, M. H.; Wrenn, B. A.; Domiguez-Espinosa, R. Production of bioenergy and biochemical from industrial and agricultural wastewater. Trends Biotechnol. 2004, 22 (9), 477−485. (30) Rodriguez, J.; Kleerebezem, R.; Lema, J. M.; van Loosdrecht, M. C. M. Modeling product formation in anaerobic mixed culture fermentations. Biotechnol. Bioeng. 2006, 93 (3), 592−606. (31) Sivagurunathan, P.; Kumar, G.; Lin, C. Y. Hydrogen and ethanol fermentation of various carbon sources by immobilized Escherichia coli (XL1-Blue). Int. J. Hydrogen Energy 2014, 39 (13), 6881−6888.
(32) Li, J. Z.; Ai, B. L.; Ren, N. Q. Effect of initial sludge loading rate on the formation of ethanol type fermentation for hydrogen production in a continuous stirred-tank reactor. Environ. Environ. Prog. Sustainable Energy 2013, 32 (4), 1271−1279. (33) Song, J. X.; An, D.; Ren, N. Q.; Zhang, Y. M.; Chen, Y. Effects of pH and ORP on microbial ecology and kinetics for hydrogen production in continuously dark fermentation. Bioresour. Technol. 2011, 102 (23), 10875−10880. (34) Ngo, T. A.; Nguyen, T. H.; Bui, H. T. V. Thermophilic fermentative hydrogen production from xylose by Thermotoga neapolitana DSM 4359. Renewable Energy 2012, 37 (1), 174−179. (35) Hung, C. H.; Cheng, C. H.; Cheng, L. H.; Liang, C. M.; Lin, C. Y. Application of Clostridium-specific PCR primers on the analysis of dark fermentation hydrogen-producing bacterial community. Int. J. Hydrogen Energy 2008, 33 (5), 1586−1592. (36) Kapdan, I. K.; Kargi, F. Review bio-hydrogen production from waste materials. Enzyme Microb. Technol. 2006, 38 (5), 569−582. (37) Lawson Anani Soh, A.; Ralambotiana, H.; Ollivier, B.; Prensier, G.; Tine, E.; Garcia, J. L. Clostridium thermopalmarium sp.nov., a moderately thermophilic butyrate producing bacterium isolated from palm wine in Senegal. Syst. Appl. Microbiol. 1991, 14, 135−139. (38) Rajhi, H.; Conthe, M.; Puyol, D.; Diaz, E.; Sanz, J. L. Dark fermentation: isolation and characterization of hydrogen-producing strains from sludges. Int. Microbiol. 2013, 16 (1), 53−62. (39) Ni, Y.; Xia, Z. Y.; Wang, Y.; Sun, Z. H. Continuous butanol fermentation from inexpensive sugar-based feedstocks by Clostridium saccharobutylicum DSM 13864. Bioresour. Technol. 2013, 129, 680− 685. (40) Ren, N. Q.; Cao, G. L.; Wang, A. J.; Lee, D. J.; Guo, W. Q.; Zhu, Y. H. Dark fermentation of xylose and glucose mix using isolated Thermoanaerobacterium thermosaccharolyticum W16. Int. J. Hydrogen Energy 2008, 33 (21), 6124−6132. (41) Shang, S. M.; Qian, L.; Zhang, X.; Li, K. Z.; Chagan, I. Themoanaerobacterium calidifontis sp. nov., a novel anaerobic, thermophilic, ethanol-producing bacterium from hot springs in China. Arch. Microbiol. 2013, 195 (6), 439−445.
G
DOI: 10.1021/acs.energyfuels.5b02143 Energy Fuels XXXX, XXX, XXX−XXX
