1. Introduction
Hydrogen can be acquired from fossil fuels and regenerable energy resources. In this context, renewable energy sources are especially important in terms of sustainability. Hydrogen has some advantages, such as being ample in nature (as it makes up about 90% of the atoms), nontoxic, environmentally friendly (producing water as a by-product) and sustainable. Recently, for environmentally friendly global energy requirements, the utilization of residual or waste biomass resources rich in carbohydrate content for biohydrogen production has become interesting [1,2,3]. Sources of lignocellulosic biomass, such as wood, grass, paper waste, domestic solid waste and agricultural residues, are the energy-source raw materials that are abundant everywhere in nature. If this resource is used for biofuel purposes, it has the advantage of reducing our dependence on non-renewable resources such as fossil fuels by minimizing greenhouse gas emissions and contributing to energy security. It has been predicted that the annual yield of lignocellulosic biomass residues in the world exceeds 220 billion tons and is equivalent to 60–80 billion tons of crude petroleum. Therefore, this low-cost and attractive raw material is an important potential for hydrogen production [4,5,6]. In the FAO [7] report, it is predicted that the request for agricultural products will increment by 35–50% between 2012 and 2050 due to the increasing population. It has been reported that this increase in demand may cause environmental negativities, such as land degradation, water pollution, water scarcity and climate change. This may include residues or wastes from agricultural activities. In areas of agricultural activity, especially crop losses may occur before harvest, during harvest stage and after harvest. This depends on pre-harvest factors, such as weather conditions, crop diversity, invasive pests and crop breeding practices. In this context, it has been estimated by FAO’s Food Loss Index (FLI) that there is a loss of approximately 14% in all foods in the post-harvest period until retail sale. In this context, environmentally friendly, sustainable and suitable biohydrogen methods should be adopted for the reduction and utilization of agricultural wastes or residues. The methods accepted as an environmentally friendly approaches for biological hydrogen production have been reported to be biophotolysis, photofermentation, dark fermentation, photo-dark fermentation and hybrid systems [8,9]. In the dark fermentation method, which uses water with various carbon-based substrates, water and carbon dioxide are produced, along with metabolites, such as acetic and butyric acid. Photofermentation and biophotolysis processes require light. Compared to photofermentation and biophotolysis processes, dark fermentation has the advantages of not requiring light and producing various metabolites [8,9]. Among these methods, dark fermentation can be considered the most hopeful method due to its ability to produce hydrogen consistently without requiring light [10]. Therefore, tons of agricultural residues or waste with high carbohydrate content can potentially be used as raw material for dark fermentation.
In order to realize biological hydrogen production, (i) organic wastes or residues and (ii) mixed microorganisms, some pretreatment methods have been applied in the literature as a single or combined method. Among these methods, the single pretreatment methods that can be listed are chemical (acid) [11] and physical (microwave and ultrasound) [12,13] methods. Combined pretreatment methods can be listed as microwave + ultrasound [13,14] and thermal + acid [15]. In biological hydrogen production, mixed bacteria were more preferred as inoculum than specific bacteria. This can be attributed to cost and accessibility. In some studies, the impression of pretreatment on biohydrogen production was investigated by applying heat pretreatments to neutralize mixed culture-containing methanogenic arcs in materials such as sludge, fertilizer and waste to be used as inoculum. In this context, mixed culture-containing sludge was subjected to heat pretreatment [16] at 80 °C for 90 min, anaerobic sludge was subjected to pretreatment [17] at 60 min and 93 °C and anaerobic sludge was subjected to heat pretreatment [18] at 121 °C for 30 min. Bansal et al. [19] examined cow manure and non-pretreated cow manure heat-treated at 100 °C for 20 min and reported that the use of pretreated inoculum resulted in significant biohydrogen production. Similarly, it was pretreated at 100 °C for 20 min, the bacteria taken from the paper mill waste were inoculated on Reinforced Clostridial Media and incubated at 37 °C to obtain the pure bacteria Bacillus licheniformis MSU AGM 2 strain and then used for biohydrogen production [20]. Lignocellulosic biomass types were generally subjected to various single or combined pretreatments. A limited number of studies have examined only the effect of mechanical grinding. The particle size of lignocellulosic biomass has been reported to affect fermentation processes [21]. Biomass size can be made more effective in fermentation processes by mechanical grinding. Thus, the compactness of lignocellulosic biomass can be reduced, and the largely crystalline state of cellulose can be transformed into an amorphous state [22,23]. There is a limited number of studies examining biohydrogen production from directly used raw materials by reducing biomass size with mechanical grinding. In this context, Yi et al. [24] examined ground Paulownia leaves for biohydrogen production as the sole carbon source. Zhang et al. [25] investigated the effect of grinding corn cobs on biohydrogen production. Although there are studies in the literature examining biohydrogen production from size-reduced raw materials subjected to various combined pretreatments, a limited variety of raw materials have been used in these studies. One of these studies was conducted by Bansal et al. [19]. The authors used vegetal wastes that were oven-dried and slurried with tap water. In a study using poplar leaves, dried and crumbled leaves were subjected to both heat (15 min at 100 °C) and acid (1% HCl) pretreatment [18]. Zhang et al. [26] applied thermal pretreatment (boiling in a beaker with dilute HCl for 30 min) to corn stalks obtained from the suburbs and ground. In the literature, there is a need for comparative data on the pretreatment and non-pretreatment of agricultural residue biomass for biohydrogen production and inferences regarding process output. The current study highlights the relative importance of filling this gap in the literature. This study focuses on the production of biological hydrogen from the raw material and the liquid extracted from the decoction-pretreated raw material. There are studies in the literature producing biological hydrogen from various agricultural residues, but there are no studies producing biohydrogen by dark fermentation from buckwheat crop residue, using mixed bacteria.
The purpose of this study is to specify the impression of pretreatment on biohydrogen production in batch bioreactors, buckwheat crop residue, direct use and use of extract liquid after extraction. In addition, a heat-pretreated mixed culture was used in this study. A microbial community analysis of this mixed culture was also performed, depending on the biohydrogen production potential of the batch bioreactor.
2. Materials and Methods
2.1. Nutritional Composition and Inoculum
The inoculum sludge used in this work was taken from the anaerobic bioreactor of the Sugar Factory biological wastewater treatment facility. Studies conducted with heat-pretreated inoculum have been reported in regard to high-hydrogen production [27]. Before the anaerobic mixed microorganism (sludge) was supplemented to the bioreactors, the sludge was heat pretreated at 97 ± 1 °C for 50 min. Thus, we aimed to increment the activity of microorganisms that support hydrogen production and inhibit or exactly eliminate the activity of methanogenic microorganisms in the content of anaerobic mixed microorganisms. The nutritional composition was prepared based on the study in the literature [28]. The nutritional composition of bioreactors is given below (Table 1).
2.2. Installation and Operation of Bioreactors
In order to produce biological hydrogen from buckwheat crop residue, laboratory-scale 120 mL volume glass anaerobic batch reactors (ABRs) were used. In order to prohibit the development of phototrophic microorganisms in ABRs, the surroundings of the glass reactors were sealed with aluminum foil tape. In the study, bioreactors were set up to consist of a 30 mL gas collection zone and a 90 mL working volume (buckwheat or buckwheat extract + pretreated mixed bacteria + nutrients). The flowchart of the study is presented in Figure 1. It has been suggested that lignocellulosic biomass types should have a particle size of less than 3 mm for effective hydrolysis and accessibility [29]. Buckwheat waste with a size of “~1 mm”, which is suitable for this size limit, was investigated for biohydrogen production in ABRs operated at pH 4.5 and pH 4.0 fed with (i) 0.40, 0.80 and 1.20 g. buckwheat/L unpretreated (raw) and with (ii) 0.40, 0.80 and 1.20 g. buckwheat extract/L pretreated (liquid extracted from decoction extraction pretreatment). In the study, thermally pretreated mixed bacteria constituted 10% of the 90 mL working volume in all bioreactors. After the addition of mixed bacteria to the bioreactors fed with raw buckwheat waste, the target remaining working volume was completed with the nutritional composition supplemented with tap water. On the other hand, after the addition of mixed bacteria to the bioreactors fed with the extracted liquid, the target remaining working volume was completed by supplementing the nutritional composition into this liquid. Decoction is an extraction application that is heat treated at 100 °C for 30 min with cold water supplementation on crumbled solid. For post-application, it is recommended to filter the solid liquid mixture by cooling at 40 °C [30]. In this work, the solid liquid mixture was filtered with filter paper when the mixture reached room temperature by making a small modification in the cooling process, and the extract liquid was used. The installation of the ABRs was completed by closing the covers, and nitrogen gas was delivered into the bioreactor for 5 min in order to remove the oxygen in the bioreactors. Finally, the bioreactors were operated in a 175 rpm shaker incubator in a dark room, at a temperature of 37 ± 1 °C. Pressure monitoring was performed with a pressure gauge connected to the bioreactor. Gas samples were taken with a glass syringe with a gas-tight stopcock and measured following sampling. Biohydrogen production, organic acids and darkroom temperature were monitored throughout the entire operation.
2.3. Analytical Methods
Organic acid samples were prepared for measurement using cellulose acetate injector filters with a pore size of 0.45 µm. Subsequently, concentrations of butyric acid, acetic acid and propionic acid were analyzed via High-Pressure Liquid Chromatography (HPLC) with the ProStar 330 PDA detector (Varian, Palo Alto, CA, USA) and Metacarb 87H (7.8 × 300 mm) column. Gas sampling was performed with a glass syringe with a gas-tight stopcock. The calibration was made with high-purity hydrogen, carbon dioxide and methane gases. Helium was used as a carrier gas. Detector, injection and column temperatures were studied at 230 °C, 200 °C and 35 °C, respectively. Gas analyses were performed using Gas Chromatography (Shimadzu, Kyoto, Japan) with a capillary colon RT-Msieve 5A (15 m, 0.53 mm ID, 50 µm DF) and Thermal Conductivity Detector (TCD). COD measurement was performed using the closed reflux method, according to standard methods [31]. The pH measurement was performed with a pH meter. Before the bioreactor setup was completed and the lids were closed, the pH was adjusted, and sampling was performed for the COD experiment. In addition, when the operation was completed, the pH was measured, and COD sampling was performed. The gas and organic acid sampling were performed at the times indicated in the figures and tables.
2.4. Microbial Community Analysis
Anaerobic batch bioreactors at different organic loading ratios and pH values were operated for the production of biohydrogen, using an anaerobic mixed microorganism with buckwheat (raw waste and extract liquid organic loading rates) residue. After the operating process was completed in all bioreactors, the microbial community in the bioreactors with high biohydrogen performance was taken. All samples taken were stored at −80 °C until the Next-Generation Sequencing (NGS) analysis was performed. In the analysis, DNA isolation and quality control of the samples were first performed to create a library. To create a library, the V3–V4 region of the 16S rRNA gene was amplified with specific primers, followed by purification. Illumina MiSeq Reagent Kit (Illumina, San Diego, CA, USA) was used. In the index PCR stage, Illumina binary indices and adapters were added using the Nextera XT index kit, and then purification was performed. Normalization was performed by measuring the concentration of the libraries created with real-time PCR and diluting them to 4nM. The normalized samples were combined via the pooling method. After the library was prepared, when each new deoxynucleoside triphosphate (dNTP) was appended via sequencing, using the synthesis method, the fluorescent radiation of the appended base was optically observed and recorded. The data produced after sequencing were transformed into raw data (FASTA format) for analysis.
Illumina library readings of the 16S rRNA gene were analyzed and processed with the QIIME2 version of the Quantitative Insights into Microbial Ecology (QIIME) software package for pretreatment and prefiltering. Read quality was controlled with FastQC, and barcodes were removed in QIIME. Following this step, readings and metadata were transferred to QIIME2 [32,33,34]. In the examples, the filtering of readings, primers and barcodes with Phred scores less than 20 and filtering of chimeric readings were performed using DADA2. The determination of taxonomic types for each sample was performed with the QIIME2 version of the standard QIIME software package.
3. Results and Discussion
In all the bioreactors, methane was not produced during the operation. Carbon dioxide production was at similar levels in all bioreactors. In the study, there was no production of hydrogen, methane or carbon dioxide in the first hours of operation. In the following hours, both hydrogen production and carbon dioxide production were detected, as shown in the figures.
3.1. The Effect of Direct Use of Biomass on Biohydrogen Production
Biohydrogen production potential was investigated at initial pH values of 4.5 and 4.0 with the use of unpretreated buckwheat residue biomass at organic loading ratios of 0.40 (on average, 79.9 mg COD/L), 0.80 (on average, 88.1 mg COD/L) and 1.20 (on average, 93.4 mg COD/L) g. buckwheat/L (Figure 2).
Gas production did not occur in the first hours of operation at pH 4.5 in bioreactors where raw biomass was directly used without pretreatment (Figure 2(A1,B1,C1)). As presented in Figure 2(A1), it was determined that hydrogen production in 0.40 g. buckwheat/L bioreactor was 1098.10−4 mL in nineteen hours and 1172.10−4 mL in twenty-eight hours. The maximum hydrogen production in this reactor was determined to be 1283.10−4 mL in thirty-one hours. At the same pH, hydrogen production increased to 2145.10−4 mL in nineteen hours and 3121.10−4 mL in twenty-eight hours in the reactor operated at 0.80 g. buckwheat/L organic loading (Figure 2(B1)). It was determined that hydrogen production decreased to 2716.10−4 mL in thirty hours. After this hour, hydrogen production showed a growing trend, and the maximum hydrogen production was determined to be 3391.10−4 mL at the forty-ninth hour. As presented in Figure 2(C1), it was determined that there was 10,658.10−4 mL, 10,821.10−4 mL and 10,740.10−4 mL of hydrogen production at eighteen, twenty-three and twenty-nine hours, respectively, in the bioreactor operated with 1.20 g. buckwheat/L and at a pH of 4.5. It was determined that there was a maximum hydrogen production of 11,749.10−4 mL at the forty-second hour, and hydrogen production decreased in the following hours.
The biological hydrogen production potential of unpretreated buckwheat biomass was also studied in the bioreactor operated at organic loading ratios of 0.40, 0.80 and 1.20 g. buckwheat/L and a pH of 4.0 (Figure 2(A2,B2,C2)). In the first hours of the study, it was determined that there was no gas production. In the bioreactor with 0.40 gr. buckwheat/L (Figure 2(A2)), the maximum hydrogen production was determined to be 2971.10−4 mL in eighteen hours. After this hour, the hydrogen production decreased and increased again. Biological hydrogen production was determined to be 2838.10−4 mL in forty-three hours and decreased over time. With 0.80 g. buckwheat/L (Figure 2(B2)), biohydrogen production in the bioreactor was 7014.10−4 mL in nineteen hours, and the maximum production was 7865.10−4 mL in twenty-seven hours. Following this hour, biohydrogen production tended to decrease. With the biological hydrogen production potential of 1.20 gr. buckwheat/L (Figure 2(C2)), 10,308.10−4 mL was reached in twenty hours, while production decreased to 9059.10−4 mL in twenty-five hours. It was determined that biohydrogen production was maximum at 11,241.10−4 mL in thirty hours, and production decreased after this hour. After the maximum hydrogen production hour, hydrogen production tended to decrease due to the pH change. Upon the completion of the study, the COD value of the effluent was determined to be in the range of 66.7–58.3 mg/L. Zhu and Béland [35] conducted a study examining the pH effect using serum bottles. In the study, it was determined that, while the pH levels were in the range of 6.3–8.0 at the beginning, the pH value decreased to the range of 4.02–5.15 in the future. In the current study, the initial pH value was 4.5 and 4.0, while the pH at the end of the operation was found to be 3.6 and 3.2, respectively. In some studies, it has been reported that the specified pH value limits the results in low-biohydrogen production. In this context, it has been reported that a pH value below 5.0 causes instability in the bioreactor, thus reducing the efficiency of hydrogen production [36]. In another work, it was reported that the inhibition of hydrogen production was caused by the pH value falling below 4.0 [37]. The current results in this work have shown that hydrogen production is not limited below the pH value limits specified in the literature and that research should be expanded. The differences in the research are mainly due to the raw material used, the pH and different microbial communities.
As given in Figure 2 (A1,A2), and as presented in Figure 2 (B1,B2), 0.40 gr. buckwheat/L when compared at 0.80 g. buckwheat/L organic loading rates according to pH 4.0 and 4.5, it resulted in high biohydrogen production at 4.0 pH operations at the same loading rates. In bioreactors operated at 1.20 g. buckwheat/L organic loading rates, pH 4.5 and 4.0, it was determined that there was maximum biohydrogen production in the reactor with pH 4.5 (Figure 2(C1,C2)). Therefore, these results showed that the pH at which the operation is carried out is substantial in the production of biohydrogen from the residue, depending on the organic loading ratio. In addition, the activity of microorganisms in the heat-pretreated anaerobic sludge content may affect the pH value. In bioreactors operated at the same pH and different organic loading ratios, the maximum biohydrogen production was determined in bioreactors with 1.20 g. buckwheat/L (Figure 2(C1,C2)). In bioreactors, as given in Figure 2(B1,A2,C2), while hydrogen production increased over time, it showed a decreasing and increasing trend again. It has been reported that the pH significantly affects hydrogen fermentation, which is due to the activity of dominant species in mixed culture [38]. The trend in the current work can be explained by the fact that the pH alteration affects the activity of the dominant species. In addition, it was determined that hydrogen production decreases over time in the operation of bioreactors. This is the result of the production of volatile fatty acids in the hydrogen production process, which can be elucidated by the decrease in the pH value.
In the literature in general, it has been reported that the pH parameter has a substantial impression on hydrogen production in both continuous feed and batch bioreactors, depending on the type of raw material. However, in many studies, the effect of the pH value differs. Li et al. [39] found that, under alkaline conditions, fermented food waste and sludge accelerated the hydrolysis phase. In another study, Kim et al. [40] examined the impression of initial pH values on the hydrogen fermentation of food wastes. In the work, it was reported that there was no substantial relationship between alkaline conditions and initial pH values. In contrast, Wang et al. [41] decomposed food waste by using sludge as an inoculum and found that acidic (pH < 4) conditions could increase hydrolysis. In another study, the application of alkaline pretreatment to raw rice straw resulted in maximum biohydrogen production [42]. While some studies have reported that alkaline conditions affect positively [39,43], some studies have reported that acidic conditions affect positively [41]. These results show that biohydrogen production is mainly specific to the pH parameter and type of raw material, so specific studies need to be expanded for these studies.
When the pH value is low, acetic acid, one of the volatile fatty acids, has been reported to be dominant [44]. In Table 2, it is seen that acetic acid produced during the hydrogen production phase at an organic loading ratio of 1.20 gr. buckwheat/L at the initial pH of 4.5 and 4.0 from the buckwheat residue not subjected to pretreatment is dominant in all fermentation systems, followed by butyric acid. The main by-products of dark fermentation have generally been reported as acetic acid and butyric acid [45,46]. These main by-products have been reported to support hydrogen production in general [27]. In the present study, it is seen that the main volatile fatty acids formed in bioreactors after batch fermentation tests are compatible with this behavior (Table 2).
Studies within the scope of biohydrogen production have generally examined acetic, butyric and propionic acids from volatile fatty acids (VFAs). Wang et al. [41] found that acetic acid was the most prevalent volatile fatty acid in food waste fermentation when the anaerobic inoculum was used with an initial pH of 4.0 and an uncontrolled pH. In another work, it was reported that biological hydrogen could be exploited as an electron donor for the propionic acid producer. Accordingly, the increase in biohydrogen consumption is the result of the increase in propionic acid production [47]. Therefore, it is important to analyze propionic acid in biological hydrogen production research. In the studies conducted by Moreno-Andrade and Buitrón [48] and Mahmoodi-Eshkaftaki et al. [49], it was reported that butyric and acetic acid were high, propionic acid was low and, thus, biohydrogen production increased. These results support the results of the current work. For the maximum performance of volatile fatty acid production, the desired results can be achieved by changing the basic operating parameters.
3.2. The Effect of the Use of Biomass Extract Fluid after Pretreatment on Biological Hydrogen Production
The organic loading rates of 0.40 (on average 98.5 mg COD/L), 0.80 (on average 102.7 mg COD/L) and 1.20 (on average 121.3 mg COD/L) g. buckwheat extract/L and biological hydrogen production potential at different initial pH (4.5 and 4.0) values were examined in batch bioreactors (Figure 3).
Pretreatment of the inoculum and the pH are critical parameters affecting biohydrogen production [50]. After pretreatment, it was determined that there was no gas production in the first hours in bioreactors where the extract liquid was operated at different organic loading rates and a pH of 4.5 (Figure 3(A3,B3,C3)). Maximum biohydrogen production: 0.40 g. buckwheat extract/L organic loading in nineteen hours, 92.10−4 mL (Figure 3(A3)); 0.80 g. buckwheat extract/L organic loading in eighteen hours, 202.10−4 mL (Figure 3(B3)); and 1.20 g. buckwheat extract/L organic loading in eighteen hours, 446.10−4 mL (Figure 3(C3)). After these hours, biohydrogen production tended to decrease steadily.
In all bioreactors, where the biological hydrogen production potential was examined at pH 4.0, with organic loading rates of 0.40, 0.80 and 1.20 g. buckwheat extract/L, it was determined that there was no gas production in the first hours of operation (Figure 3(A4,B4,C4)). Maximum biohydrogen production: 0.40 g. buckwheat extract/L organic loading in nineteen hours, 136.10−4 mL (Figure 3(A4)); 0.80 g. buckwheat extract/L organic loading in eighteen hours, 158.10−4 mL (Figure 3(B4)); and 1.20 g. buckwheat extract/L organic loading in eighteen hours, 391.10−4 mL (Figure 3(C4)). Hours after the biohydrogen production was at its maximum, biological hydrogen production tended to decrease steadily. Upon completion of the study, the COD value of the effluent was determined to be in the range of 89.7–79.6 mg/L.
At the same organic loading rates, the effect of the pH values on biohydrogen production was examined. As given in Figure 3(A3,A4), maximum biohydrogen production at a pH of 4.0 was determined in bioreactors with organic loading ratios of 0.40 g. buckwheat extract/L. In Figure 3(B3,B4); the maximum biohydrogen production was found to be at pH 4.5 in bioreactors with organic loading rates of 0.80 g. buckwheat extract/L. At this pH (4.5), as presented in Figure 3(C3), the bioreactor with an organic loading ratio of 1.20 g. buckwheat extract/L had a higher biohydrogen production than the reactor operated at pH 4.0. Among the bioreactors operated at organic loading ratios of 0.40, 0.80 and 1.20 g. buckwheat extract/L and pH 4.5, maximum biohydrogen production was determined in the bioreactor with an organic loading rate of 1.20 g. buckwheat extract/L (Figure 3(A3,B3,C3)). Among the bioreactors operated at the same organic loading rates and pH 4.0, biohydrogen production was found to be maximum in the bioreactor with an organic loading ratio of 1.20 g. buckwheat extract/L (Figure 3(A4,B4,C4)). In the work handled by Ghimire et al. [42] which examined the impression of pH value on biohydrogen production from waste biomass in batch reactors, the initial pH value of food waste was examined in the range of 4.5–7.0, and the maximum biohydrogen production was determined at 4.5 pH. In other studies that examined the impact of initial pH values on hydrogen production, maximum hydrogen production was achieved at an initial pH of 9.0 [51] from sucrose and at an initial pH of 7.21 [52] from food waste. In this present work, while the initial pH value was 4.5 and 4.0, it was found that the pH decreased to close to 3.4 at the end of the study. When these studies and the current study were evaluated, it was determined that both the pH and organic loading rate significantly affected biohydrogen production depending on the raw material and inoculum used. In the work handled by Mota et al. [53], hydrogen production from sucrose via dark fermentation was investigated in the reactor operated at 30 °C without pH control. In the work, it was determined that hydrogen production took place during the tests, and the pH value of the outlet water was 2.8 on average. In this study, hydrogen production was detected, contrary to the literature data reporting inhibition at pH values below 4.0 in biohydrogen production studies with dark fermentation.
The results of the analysis of organic acids in bioreactors with an organic loading ratio of 1.20 g. buckwheat extract/L, operated at the initial pH of 4.5 and 4.0, obtained from pretreated buckwheat biomass are presented in Table 3. In bioreactors, acetic acid was found to be dominant, followed by butyric acid.
Similar to the present study, in a study with heat-pretreated inoculum, the amount of high organic acids was determined as acetic and butyric acid, respectively. These results show that there is a suitable microenvironment that supports hydrogen production [27]. In another work, Cruz-López et al. [54] examined the production of biohydrogen from food industry wastewater in an upflow anaerobic sludge blanket reactor and reported that acetic (approximately 590 mg/L) and butyric (approximately 450 mg/L) acids account for most of the production of volatile fatty acids. In the work conducted by Alexandropoulou et al. [55], it was reported that low propionic acid production may be an indicator that the hydrogen production process is efficient. In the work handled by Jankowska et al. [56], the effect of fermentation using anaerobic mixed culture on the production of volatile fatty acids was examined. In the study, it was reported that propionic acid was the main product instead of acetic acid at a pH of 4.0. In the present work, it was determined that the propionic acid concentration was very low in bioreactors operated at pH 4.5 and 4.0, as given in Table 3. These results show that biohydrogen production was based on the nature and source of the inoculum, raw material and bioreactor operating conditions.
3.3. Microbial Community Content
Although the impression of parameters such as pH, raw material concentration and temperature on biohydrogen production has been examined in some studies, a limited number of studies have examined the role of the mixed microbial community on biological hydrogen production. In the present study, species-level ratios were determined via a taxonomic content analysis. Since the maximum biohydrogen production was reached at pH 4.5 and 4.0 at an organic loading rate of 1.20 g. buckwheat/L in bioreactors where raw biomass was directly used without pretreatment, the microbial community was taken in the content of these bioreactors for analysis. In the results, as given in Figure 4(C1), the most dominant species in hydrogen production of total bacterial populations in the bioreactor operated at pH 4.5 is Hathewaya histolytica, with a rate of 17.08%. This is followed by Clostridium butyricum, with a rate of 15.37%; and Levilinea saccharolytica, with a rate of 10.12%. In the study, since microorganisms below 2% could not be determined, they were specified as “others”, and others were determined as 24.99%. In the reactor operated at pH 4.0, Clostridium butyricum was found to be the dominant bacteria in hydrogen production, with a rate of 17.05%; Hathewaya histolytica, 7.62%; [Clostridium] populeti, 7.58%; and Levilinea saccharolytica, 6.72%, as presented in Figure 4(C2).
Considering that biohydrogen production is maximum in bioreactors where extract liquid is used after pretreatment, at pH 4.5 and 4.0, at organic loading rates of 1.20 g. buckwheat extract/L, the biohydrogen production was analyzed by taking into account the microbial community in these bioreactors. In the bioreactor operated at pH 4.5, the dominant bacteria in hydrogen production were determined to be Clostridium butyricum, 25.06%; Levilinea saccharolytica, 14.80%; Clostridium swellfunianum, 11.23%; and Clostridium tertium, 7.41% (Figure 5(C3)). In addition, microorganisms below 2% were detected as 31.80% of “others”. As given in Figure 5(C4), in the reactor operated at pH 4.0, Clostridium tertium was determined as 30.62%, Levilinea saccharolytica as 15.94%, Clostridium swellfunianum as 6.37%, Clostridium butyricum as 5.25% and others as 34.36%.
In the analysis results, the bacterial composition was determined at rates of 2.0% and above. The results showed that bacteria are the main microorganism in biohydrogen production. Rafrafi et al. [57] reported that sub-dominant species in mixed culture may have a substantial effect on fermentative hydrogen production. In the present study, the detection of “others” at rates of 22% and above indicates that low-abundance microorganisms may play a key role in biological hydrogen production.
Hydrogen-manufacturing bacteria are commonly known as Clostridium species [58,59]. In this context, Clostridium Butyricum [60,61,62] has especially been used in biohydrogen production research. Most of the bacteria that carry out hydrogen production can grow in a wide pH range. In addition, it has been reported that these bacteria grow very quickly compared to methane-producing bacteria [35]. Due to their propionate production, Selenomonas ruminantium, Butyrivibrio fibrisolvens and Megasphaera elsdenii bacteria can be considered hydrogen consumers [63,64]. The presence of such propionate-producing bacteria can limit the growth of hydrogen-manufacturing bacteria and the production of biological hydrogen. In the present study, too-low propionic acid production can be explained by the absence or very limited presence of propionic acid-producing bacteria.
Hathewaya histolytica was found to be the most dominant bacteria in the reactor operated at pH 4.5, and Clostridium butyricum was found to be the most dominant bacteria in the reactor operated at pH 4.0, where raw biomass was directly used without pretreatment. Clostridium butyricum was found to be the most dominant bacteria in the reactor operated at pH 4.5, and Clostridium tertium was found to be the most dominant bacteria in the reactor operated at pH 4.0, where the extract liquid was used after pretreatment. In the study handled by Kim et al. [65] in which the effect of the initial pH value on the microbial community was examined, it was determined that the initial pH value had no substantial impression. The reason for this is that the microorganisms in the food waste were used in the study, and no inoculum was used for fermentation. The impression of the initial pH value on hydrogen production was also examined by Tang et al. [66]. In the study, it was determined that the significant change in the initial pH value did not cause a substantial change in the dominant microbial population during biohydrogen production by conducting a microbial community analysis. In another study, it was reported that the pH of the operating conditions, the metabolic pathways and the entire population of bacteria present in hydrogen production affected the pH parameter [67]. The results of the analysis in this current work showed that there was no substantial change in the dominant microbial population ratios during biohydrogen production, even if there was a change in the pH. In the current study, when the analysis results of bioreactors fed with the same raw material but operated at different pH values were examined, it was determined that there was no significant change in the dominant microbial population ratio according to the pH values. In addition, after using such residues or wastes directly, it was concluded that bioreactor operations at pH 4.5 and 4.0 are suitable for the activity of bacteria that perform hydrogen production.
4. Conclusions
For environmental pollution reduction and clean energy, using agricultural residues or wastes is an attractive approach. In the study, the direct use of raw buckwheat biomass without pretreatment and the use of extract liquid after pretreatment of buckwheat biomass were examined in batch bioreactors according to different organic loading ratios and initial pH values. It has been determined that biohydrogen production occurs in all operating conditions of bioreactors. The maximum biohydrogen production was determined to be 11,749.10−4 mL at 1.20 g. buckwheat/L and 446.10−4 mL at 1.20 g. buckwheat extract/L at a pH of 4.5. The results of the microbial community analysis showed that pH differences did not cause a substantial change in the species-level ratios of bacteria. In bioreactors using biomass directly, Hathewaya histolytica 17.08% and Clostridium butyricum 17.05% at pH values of 4.5 and 4.0, respectively; in bioreactors using biomass extract liquid, Clostridium butyricum 25.06% and Clostridium tertium 30.62% were found to be the most dominant bacteria at pH values of 4.5 and 4.0, respectively. Each of these bacteria can be used as specific bacteria for the purpose of biohydrogen production. With this study, it was determined that buckwheat residue can be used successfully in biological hydrogen production.
The author confirms that the data supporting the findings of this study are available within the article.
The author would like to thank METU Molecular Biology and Biotechnology R&D Center.
The author declares no conflicts of interest.
Footnotes
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Enlarge this image.Figure 2. Hydrogen gas production in bioreactors fed with 0.40 (A1,A2), 0.80 (B1,B2) and 1.20 (C1,C2) g buckwheat/L.
Enlarge this image.Figure 3. Hydrogen gas production in bioreactors fed with 0.40 (A3,A4), 0.80 (B3,B4) and 1.20 (C3,C4) g buckwheat extract/L.
Enlarge this image.Figure 4. Species level ratios in the bioreactor where buckwheat residual biomass is used directly.
Enlarge this image.Figure 5. Species-level ratios in the bioreactor using buckwheat residue extract fluid.
Nutritional composition in bioreactor.
| Nutritional Composition | Unit (mg/L) |
|---|---|
| KH2PO4 | 250 |
| NH4Cl | 2500 |
| CaCl2 · 2H2O | 500 |
| MgSO4 · 7H2O | 320 |
| MnCl2 · 4H2O | 30 |
| NiSO4 | 32 |
| ZnCl2 | 23 |
| CoCl2 · 6H2O | 21 |
| FeCl3 · 6H2O | 20 |
| CuCl2 · H2O | 10 |
| Na2MoO4 · 2H2O | 14.4 |
Organic acid analysis in bioreactors using buckwheat residual biomass directly.
| pH–Hour | Acetic Acid | Butyric Acid | Propionic Acid |
|---|---|---|---|
| 4.5–18 | 1.647 ± 0.001 | 0.836 ± 0.001 | 0.022 ± 0.003 |
| 4.5–23 | 1.722 ± 0.004 | 0.838 ± 0.000 | 0.034 ± 0.000 |
| 4.5–42 | 1.775 ± 0.002 | 0.844 ± 0.001 | 0.041 ± 0.001 |
| 4.5–66 | 1.535 ± 0.016 | 0.814 ± 0.004 | 0.018 ± 0.004 |
| 4.5–73 | 1.338 ± 0.000 | 0.747 ± 0.000 | 0.006 ± 0.000 |
| 4.0–20 | 1.560 ± 0.004 | 0.990 ± 0.001 | 0.036 ± 0.000 |
| 4.0–25 | 1.302 ± 0.001 | 0.894 ± 0.003 | 0.017 ± 0.001 |
| 4.0–30 | 1.790 ± 0.002 | 1.015 ± 0.001 | 0.052 ± 0.001 |
| 4.0–42 | 1.788 ± 0.001 | 1.022 ± 0.002 | 0.053 ± 0.001 |
| 4.0–48 | 1.693 ± 0.009 | 1.004 ± 0.001 | 0.046 ± 0.004 |
Organic acid analysis in bioreactors using buckwheat residue extract fluid.
| pH–Hour | Acetic Acid | Butyric Acid | Propionic Acid |
|---|---|---|---|
| 4.5–18 | 1.114 ± 0.009 | 0.244 ± 0.001 | 0.038 ± 0.001 |
| 4.5–25 | 0.980 ± 0.001 | 0.255 ± 0.004 | 0.020 ± 0.001 |
| 4.5–28 | 0.835 ± 0.001 | 0.236 ± 0.001 | 0.008 ± 0.000 |
| 4.0–18 | 0.935 ± 0.001 | 0.237 ± 0.001 | 0.020 ± 0.001 |
| 4.0–25 | 0.790 ± 0.001 | 0.221 ± 0.000 | 0.013 ± 0.000 |
| 4.0–28 | 0.732 ± 0.003 | 0.237 ± 0.001 | 0.005 ± 0.000 |
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