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Volatile fatty acids production from saccharification residue from food waste ethanol fermentation: Effect of pH and microbial community
Bioresource Technology 292 (2019) 121957
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Volatile fatty acids production from saccharification residue from food waste ethanol fermentation: Effect of pH and microbial community
T
Yong Jina,b, Yujia Lina,b, Pan Wangc, Runwen Jina,b, Ming Gaoa,b, Qunhui Wanga,b, ⁎ Tien-Chin Changd, Hongzhi Maa,b, a
Department of Environmental Engineering, University of Science and Technology Beijing, 100083, China Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, Beijing 100083, China c Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry, Beijing Technology and Business University, Beijing 100048, China d Institute of Environmental Engineering and Management, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Rd., Taipei 106, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Food waste Anaerobic fermentation VFAs pH Community analysis
In this study, residue from saccharification and centrifugation of food waste ethanol fermentation was used as substrate to produce volatile fatty acids. The effects of different pH (5.5, 6.5, and uncontrolled) on the VFAs concentration, composition, acidogenic efficiency and microbial community distribution were investigated. The results showed that the highest concentration of VFAs was 267.8 ± 8.9 mg COD/g VS at pH of 6.5, and the highest percentage of butyric acid (79.8%) was followed by propionic acid and acetic acid at the end of the reaction. Microbial analysis showed that the contents of Vagococcus and Actinomyces increased, while the contents of Bacteroides and Fermentimonas decreased during anaerobic fermentation. The comparative high pH induced the accumulation of butyric acid. This study provides a new idea for the step anaerobic fermentation of food waste to produce alcohol and acid simultaneously.
1. Introduction Food waste, with high water content, high organic matter, high salt content and perishability, has drawn many researches’ interest on its resource utilization (Ren et al., 2018). Compared with general treatments, such as incineration, landfill, composting, the most effective way is anaerobic fermentation, which produces acid or methane (Zhou et al., 2018). However, the Volatile Fatty Acids (VFAs) production has a shorter time than methane fermentation (Ma and Liu, 2019). VFAs produced by the fermentation of food waste can be used as a carbon source for nitrogen and phosphorus removal in wastewater treatment (Wang et al., 2014b), as a raw material for producing biodegradable plastics (Jiang et al., 2014), and vinyl acetate (Jiang et al., 2013). In addition, VFAs can be further used by microorganisms to produce hydrogen (Chen et al., 2013b), methane and biodiesel (Pham et al., 2015), PHA and electricity (Chen et al., 2013a), and for chain elongation (Roghair et al., 2018), etc. Although VFAs production can achieve energy recovery of food waste, the separate fermentation of food waste under high load may cause problems such as excessive Na+ concentration, excessive accumulation of organic acids, and rapid pH decrease due to excessive
⁎
acidification rate (Gelegenis et al., 2007). Besides food waste, sludge also has great value for energy recovery (Li et al., 2019a). But it is easy to be alkaline by hydrolysis alone, resulting in excessive pH, high ammonia nitrogen concentration, high heavy metal content and pathogen inhibition, which may adversely affect the fermentation process (Iacovidou et al., 2012). Therefore, some researchers have proposed the co-fermented of food waste and sludge to produce VFAs, which could not only make efficient use of organic substances in food waste and sludge, but also reduce the pollution (Chen et al., 2013b; Feng et al., 2015; Wu et al., 2016). During anaerobic fermentation, the fermented products and their composition are affected by various factors. As an important environmental factor, pH can not only affect the activity of microbial enzymes, but also determine the presence of VFAs (Garcia-Aguirre et al., 2017). Many researchers studied the effect of pH on the hydrolysis and acidification of organic solid waste by dynamically adjusting the pH value. Some researchers believed that anaerobic fermentation under alkaline conditions could inhibit the activity of methanogens, which was conducive to the accumulation of VFAs (Yan et al., 2010; Yang et al., 2014; Yu et al., 2008; Zhang et al., 2009). However, in the practical application of fermentation engineering, anaerobic fermentation will
Corresponding author. E-mail address: [email protected] (H. Ma).
https://doi.org/10.1016/j.biortech.2019.121957 Received 1 July 2019; Received in revised form 2 August 2019; Accepted 3 August 2019 Available online 07 August 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 292 (2019) 121957
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gradually reduce the pH to acidic. If it is adjusted to alkaline conditions, Na+ will accumulate and inhibit the activity of acidogenic microorganisms, resulting in a decrease in subsequent acid production capacity. In addition, it would not be economically feasible to adjust the pH to alkaline conditions (Jankowska et al., 2017). Therefore, it is more economical and reasonable to control pH to optimize acid production under weakly acidic conditions. Jiang et al tested the effect of anaerobic digestion of food waste at pH 5, 6, 7 and without pH control under medium temperature conditions (35 °C) (Jiang et al., 2013). The results showed that when the pH was 6, the acidogenic efficiency of food waste was the best. Wang and Yin et al. used aerobic and anaerobic activated sludge as inoculum to study the effect of pH (4, 5, 6) on the yield of VFAs using food waste as a substrate (Wang et al., 2014a). The results showed that pH 4 was more favorable for the hydrolysis of organic matter than other pH values, and the best pH for VFAs production was at pH 6. However, there were few researches on microbial community changes at different stages under faintly acid conditions. The organic matter in the food waste was rich and complicated. In order to further utilize the various organic substances in a high-efficiency way, researchers used the stillage of food waste to produce ethanol or lactic acid, and the residue was used for composting or sanitary landfill (Ma et al., 2016). However, this method cannot fully utilize the organic substances in food waste, the remaining saccharification residue still contained a small amount of sugar and protein, and there existed little research on how to treat the residue. The purpose of this study is to further utilize the residue from saccharification and centrifugation of food waste ethanol fermentation (RSCFW). Co-fermentation was carried out by adjusting different pH and inoculating anaerobic sludge with acclimation to investigate the effect of pH on VFAs production, composition and microbial community structure during anaerobic fermentation.
Table 1 Characteristics of RSCFW and Seeding sludge.
2. Materials and methods
stirring was controlled by a programmable logic controller (PLC) at 250 r/min, and the pH was adjusted with 3 mol/L HCl solution and 3 mol/L NaOH solution. The values were 5.5 and 6.5, respectively, and the anaerobic environment was maintained during the whole fermentation process. The fermentation time was 72 h and samples were obtained every 12 h.
Parameters
RSCFW
Seeding sludge
pH TS/% VS/% C (%) N (%) Carbohydrate (%) Protein (%)
6.3 ± 0.1 26.3 ± 2.3 25.1 ± 2.1 46.1 ± 3.2 3.6 ± 0.2 48.9 ± 4.4 15.9 ± 1.6
6.5 ± 0.1 8.4 ± 0.4 5.0 ± 0.1 28.73 ± 2.3 3.9 ± 0.2 64.2 ± 5.9 11.2 ± 1.1
Note: the pH in RSCFW was measured after adding deionized water by 2:1.
Fig. 1. Variation of VFAs and ethanol concentration at different pH value.
2.1. Materials 2.1.1. Residue from saccharification and centrifugation of food waste (RSCFW) The food waste was taken from a canteen in a university in Beijing. After preliminary sorting, crushing and passing through a 3-mm sieve, it was diluted with water by a mass ratio of 2:1 (food waste: water) and placed in a water bath. Saccharification enzyme with the dose of 100 U/ g was added, the saccharification was carried with rotation 60 r/min at 60 °C in water bath for 6 h, and then centrifuged at 12,000 r/min for 10 min, the liquid and residue of food waste were detained (Ma et al., 2016).
2.3. Analytic methods All of the samples were centrifuged for 10 min at 12,000 r/min and then the supernatants were filtered through a 0.45 µm membrane. All the tests were carried out in parallel triplicates and the average values were determined. 2.3.1. VFAs and ethanol test VFAs and ethanol were measured by gas chromatograph (GC-2010 plus, Shimadzu, Japan) equipped with a capillary column (DB-FFAP) and a flame ionization detection (FID). The temperature rise procedure was as follows: initial temperature of 60 °C for 2 min, increased to 100 °C at 20 °C/min, held for 1 min, increased to 170 °C at 10 °C/min, maintained for 1 min, and raised to 210 °C at 20 °C/min for 2 min. The inlet (SO1 PL1) temperature, FID temperature, and injection volume were 220 °C, 240 °C, and 0.4 µL, respectively (Yu et al., 2018).
2.1.2. Seeding sludge with acclimation The sludge was taken from a domestic sewage treatment plant in Beijing, first concentrated at ambient temperature for 24 h, and the precipitated sludge was treated in a water bath at 105 °C for 2 h in order to kill methanogens. Then it was transferred to a 500-mL shake flask and added to the food waste (5% g VS/g) for anaerobic acclimate for two weeks. When the pH went down from 7.8 to about 6.5 (Liu et al., 2016), the anaerobic sludge was centrifuged at 4000 r/min for 10 min to obtain acclimation sludge. The basic properties of saccharified residue and acclimation sludge were shown in Table 1.
2.3.2. Other analytical methods Soluble Chemical Oxygen Demand (SCOD) was measured using standard methods (APHA, 2012). TS was calculated from the dry organic waste from the filter after heating in an oven at 105 °C, and VS was measured after the dry organic waste was burned in a muffle furnace. The pH was measured by a pH probe (pHSJ-3F, China). The acidification yield (ηa ) (Eq. (1)) was calculated as the ratio of the cumulative VFAs and final concentration of SCOD in the leachate
2.2. Experimental setup The RSCFW and seeding sludge were mixed at a mass ratio of 2.5:1 (Volatile Solid (VS) ratio) and Total Solid (TS) of 10%, then put into a reaction apparatus, and then was added 2-bromoethanesulfonic sodium salt (BES, Adamas, ≥98%) (50 mmol/L) (Jin et al., 2019). The temperature was controlled by the heating device at (37 ± 1) °C, the 2
Bioresource Technology 292 (2019) 121957
Y. Jin, et al.
(Jankowska et al., 2017):
ηa =
VFAs × 100% SCOD
(1)
The VFAs/SCOD indicated how many soluble substances were converted into VFAs by fermentation. The higher the ratio, the higher the VFAs conversion rate is. 2.4. Microbial community analysis 2.4.1. DNA extraction and PCR amplification Microbial DNA was extracted from V4 to V5 samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to manufacturer’s protocols (Yan et al., 2019). The V4-V5 region of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min) using primers 515F ( 5′-GTGCCAGCMGCCGCGG-3′), 907R (5′-CCGTCAATTCMTTTRAGTTT-3′), where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate of 20-μL mixtures containing 4 μL of 5× FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA, for amplification. Finally, the PCR products were sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). 2.4.2. Illumina MiSeq sequencing Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions and quantified using QuantiFluor™-ST (Promega, USA). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA556916). 2.4.3. Processing of sequencing data Raw fastq files were demultiplexed, quality-filtered using QIIME with the following criteria: (1) 300 bp reads were truncated at any site receiving an average quality score < 20 over a 10 bp sliding window, discarding the truncated reads that were shorter than 50 bp; (2) no ambiguous bases were allowed, and no mismatches were allowed in the primer sequence. Operational units (OTUs) were clustered with 97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/ ), and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the Silva (SSU115) 16S rRNA database using confidence threshold of 70%. The general differences of microbial community structure were evaluated by principal component analysis (PCA) in Canoco 5.0 (Microcomputer Power, USA) using the relative abundances of the whole microbial communities. 3. Results and discussion 3.1. Effect of pH on the VFAs concentration and composition (1) VFAs and ethanol concentration In the process of hydrolysis and acidification, microorganisms secrete extracellular hydrolase, which hydrolyzes complex macromolecular organic substances (proteins, polysaccharides, oils, etc.) into small molecular organic substances (such as monosaccharides, amino acids, glycerol and long-chain fatty acids). Acidogenic microorganisms used these small molecules to produce pyruvic acid, lactic acid, etc., which were then converted to VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) or other products (Yin et al., 2016). The results of acid production of saccharified residue
Fig. 2. Variation of VFAs composition at different pH value.
3
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Table 2 VFAs composition under different pH value at the end of fermentation (%). pH
Acetic acid
Propionic acid
Isobutyric acid
Butyric acid
Isovaleric acid
Valeric acid
5.5 6.5 Uncontrolled
6.9 4.6 20.7
7.7 9.4 12.7
1.1 1.5 1.9
81.0 79.3 61.4
2.0 3.5 3.3
1.3 1.7 0
uncontrolled pH, respectively. Jiang et al found that when pH was 6, the concentration of VFAs reached a maximum of 37.60 g/L, 9 times of those without pH control (Jiang et al., 2013). In addition, Wang et al. found that pH control at 5.0 to 6.5 favored hydrolysis and acidification, and the highest VFAs yield (0.918 g/g VSSremoval) was obtained at pH 6 (Wang et al., 2014a). At the end of the fermentation, the concentrations of VFAs were 232.5 ± 11.3, 232.4 ± 11.2, and 98.7 ± 9.3 mg COD/g VS, respectively. Ren et al. (2011) observed that acid-producing bacteria were inhibited at pH < 4.0, and methanogens were activated when pH > 6.5 (Yuan et al., 2006). It could be seen from Fig. 1 that when the pH was controlled, the initial VFAs concentration was different, indicated that the initial pH might affect the dissolution of the substrate (Yang et al., 2014). At the beginning of the reaction (0–12 h), the concentration of VFAs decreased rapidly, indicated that the initial pH change would affect the activity of acid-producing bacteria. Then VFAs increased slowly, indicating that the acid-producing bacteria adapted to the pH environment. At the end of fermentation, the concentration of VFAs at pH 5.5 still increased, indicating that the pH condition was suitable for the growth of acid-producing bacteria (Nzeteu et al., 2018). In addition to VFAs, ethanol was also produced, albeit at a lower level. There were many studies on the production of ethanol by anaerobic fermentation of food waste. However, the substrate in this study was RSCFW, which produced low ethanol concentration by co-fermentation with sludge. When the pH was 6.5, the concentration of ethanol reached the highest (27.9 ± 0.7 mg/g VS). There might be the following reasons, the main component of RSCFW was protein (nitrogen source), but ethanol fermentation mainly used sugar, so ethanol production was very low.
Fig. 3. Variation of SCOD and VFAs/SCOD at different pH value. Table 3 Samples information for bacterial community structure analysis. Name
Sample source
Sampling time (h)
1_solid 2_mixed 3_fermented
RSCFW RSCFW & seeding sludge Output in the reactor at pH 6.5
0 0 72
(2) VFAs composition According to the composition of products, anaerobic fermentation could be divided into different fermentation types, such as butyric acid fermentation, propionic acid fermentation, mixed acid fermentation, and so on. By investigating anaerobic digestion of food waste under different acidic conditions for VFAs production, there were different results (Lee et al., 2014). Min et al. (2005) found that the maximum propionate fraction (80%) was observed at pH 6.5 in a continuous reactor. Bengtsson et al. (2008) reported that acetic and propionic acids were the main components during the acidogenic fermentation of different paper mill wastewaters at pH 6.0. Dong-Hoon et al. (2011) found that the production of butyrate was highest at pH 8.0 comparing with other pH at 5.0, 6.0, 7.0 and 9.0. Jiang et al. (2013) found that when the pH was at 5.0 or uncontrolled, acetate was the dominant product, followed by butyrate, propionate, and valerate; and when the pH was at 6.0 and 7.0, butyrate became the main product. Wang et al. (2014a) found that butyrate was the primary product under pH 5.0 with the percentage of butyrate above 80%. The difference among these studies is mainly due to the different choices of substrates and reactors. In our study, we explored the composition of the product under pH control with RSCFW as the substrate (Fig. 2). As shown in Fig. 2, the products mainly contained acetic acid, propionic acid, and butyric acid under different pH conditions. When the pH was 5.5 and 6.5, the acetic acid ratio reached the highest at the 12th hour, followed by propionic and butyric acids. At the end of fermentation, the order of these three main products were converse. When
Fig. 4. The bacterial community analysis from different sample source at phylum level.
were shown in Fig. 1. It could be seen that the concentration of VFAs without pH control was the lowest in 0–36 h. Those with pH of 5.5 decreased continuously, while those with pH of 6.5 decreased first and then rose rapidly. The highest concentration of 267.8 mg COD/g VS was obtained with pH of 6.5 at the 36 h, 3 and 10 times higher than those pH of 5.5 and 4
Bioresource Technology 292 (2019) 121957
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Table 4 Phylogenetic classification of the 16S rRNA gene sequences. Phylum
Class (%)
Solid
Mixed
Fermented
Genus (%)
Solid
Mixed
Fermented
Firmicutes
Bacilli
98.58
7.01
35.92
Clostridia
0.0021
32.16
33.59
Weissella Bacteroides Clostridium_sensu_stricto_1 Vagococcus Bacillus Fermentimonas Actinomyces Terrisporobacter Turicibacter Romboutsia
59 0 0.0021 0.011 36 0 0 0 0 0
0.028 40 21 5.5 0.19 12 1.8 3.6 3.7 1.7
0.011 12 23 34 0.57 5.2 9.1 3.6 3.4 1.6
Globicatella norank_f_Rikenellaceae Norank_o_Chloroplast
0 0 1.4
1.2 1.9 0
0.93 0.0081 0
Bacteroidetes
Erysipelotrichia
0
3.74
3.45
Bacteroidia
0
54.36
17.50
Actinobacteria
Actinobacteria
0
2.02
9.37
Ruminococcaceae_NK4A214_group
0
0.20
1.0
Cyanobacteria
Oxyphotobacteria
1.36
0
0
Leuconostoc
1.1
0
0
Others
Others
0.06
0.71
0.16
Others
3.0
7.8
5.5
It could be seen that when the pH was 6.5, the concentration of SCOD decreased rapidly in the first 12 h and then increased slowly. As for pH 5.5 and uncontrolled, the concentration of SCOD tended to be stable. At the end of fermentation, the concentration of SCOD with uncontrolled was highest (492.75 ± 6.5 mg/g VS). The result showed that the solubility of SCOD was higher with pH uncontrolled, which was contrary to (Jiang et al., 2013). It was probably due to the rapid acidification which resulted in the death of most of the bacteria in the inoculum and the dissolution of the organic matter in the cell, resulting in the higher concentration of SCOD (Warnecke and Gill, 2005). On the other hand, when the pH was 6.5, VFAs/SCOD was significantly higher than the other two groups, and the highest VFAs conversion rate reached to 61.6 ± 2.5% at 36 h, less than those without pH control (83.46%) (Wu et al., 2016), this was because the organic matter in our substrate was much less. At the end of fermentation, the VFAs/SCOD at pH 5.5 was higher than pH 6.5, it indicated that the strains with pH 5.5 might adapt to the acid environment (Tang et al., 2016). And this can also explain the phenomenon of the continuous increase of VFAs at pH 5.5 in Fig. 1.
Fig. 5. The bacterial community analysis from different sample source at genus level.
3.3. Microbial community analysis
the pH was uncontrolled, although the butyric acid ratio continued to increase, the acetic acid content was higher than that of the other two groups. Moreover, Table 2 showed the VFAs composition under different pH at the end of fermentation. It can be seen that acetic acid fermentation was the main reaction at the first 24 h (pH 5.5–36 h), and then butyric acid production gradually increased with the change of dominant strain. Propionic acid concentration decreased slightly and the overall trend was not obvious. At the end of fermentation, the ratio of butyric acid was 81.0%, 79.3%, and 61.5% under different pH conditions. The results showed that under the pH 5.5–6.5, the dominant strain was for butyric acid production. This may be due to the chain elongation under pH control, i.e. the acetic acid of two carbons elongated to butyric acid of four carbons (Suo et al., 2018).
During the anaerobic fermentation, the microbial activity would be affected by different treatment conditions. He et al. (2019) studied the changes of microbial community in the production of VFAs from food waste by high concentration of NaCl. Wu et al. (2016) investigated the effect of uncontrolled pH on microbial community in the co-fermentation of food waste and excess sludge to produce VFAs. Li et al. (2019b) studied the variation of microbial community at pH 6, 7, and 8. However, few studies have concentrated on the changes of microbial community structure at pH 5.5–6.5. Therefore, it was necessary to further analyze the variation of substrate, inoculum and fermented products under pH control. Sample information of bacterial community structure in this study was shown in Table 3. As shown in the Fig. 4 about the phylum level analysis, all the dominant bacteria were Firmicutes, accounting for 98.6%, 42.9%, and 73.0%, respectively. Firmicutes were the dominant hydrolyzed bacteria which were capable of degrading sugars, fats, and proteins for acid production (Dareioti and Kornaros, 2014). In the mixed stage, another dominant group was Bacteroidetes (54.4%), and finally it decreased to 17.5% in the fermented stage. Bacteroidetes was a major bacterium for methane production, and it was inhibited with the BES addition (Yu et al., 2018). The Actinobacteria, increased slightly from 2.0% to 9.4%, could promote the hydrolysis of cellulose and the decomposition of glucose (Kim et al., 2010). The increase in the abundance of
3.2. Effect of pH on the acidogenic efficiency In the acidogenic fermentation, insoluble substances in RSCFW were gradually hydrolyzed into soluble proteins, fats, etc. And they would further be fermented into VFAs, H2, and CO2 with the microorganisms, thus, the SCOD of RSCFW would be changed. Besides, it was necessary to study the acidogenic efficiency by calculating the VFAs/SCOD. The variation of SCOD and VFAs/SCOD at different pH are shown in the Fig. 3. 5
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Actinobacteria also indicated the decomposition of organic matter in the pH 6.5 could better promote the acid production process. In order to further demonstrate the functional evolution of the bacterial microbial community, the bacteria were analyzed at the genus level, and the composition and comparison of major species were shown in Table 4 and Fig. 5, respectively. It could be seen from Fig. 5 that the major seven genera were Weissella, Bacteroides, Clostridium_sensu_stricto_1, Vagococcus, Bacillus, Fermentimonas, and Actinomyces, they belonged to the class Bacilli and Clostridia (Table 4). In the RSCFW, the dominant genus Weissella (59%) belonged to the Firmicutes, producing CO2 from carbohydrate metabolism with lactic acid and acetic acid as major end products from sugar metabolism (Fusco et al., 2015). In the mixed stage, the dominant genus changed from Weissella to Bacteroides (40%), which was a type of anaerobe within the Bacteroidetes phylum, and succinic acid was the major fatty acid byproduct of Bacteroides metabolism (Rotstein et al., 1985). In the fermented stage, the major genus was the Vagococcus, which was mainly producing lactic acid as the end product (Wilhelm and Brian, 2014). Compared the change of dominant bacteria between the mixed and fermented stage, it could be found that the Bacteroides and the Fermentimonas decreased from 40% to 12% and 12% to 5.2%, respectively. Conversely, the Vagococcus and the Actinomyces were increased from 5.5% to 34% and 1.8% to 9.1%, respectively. With the pH 6.5, more lactic acid was produced in the first stage, and then it would be degraded, so there was more butyric acid produced from acetic acid, and there would be a chain elongation in the next stage.
Garcia-Aguirre, J., Aymerich, E., González-Mtnez de Goñi, J., Esteban-Gutiérrez, M., 2017. Selective VFA production potential from organic waste streams: assessing temperature and pH influence. Bioresour. Technol. 244, 1081–1088. Gelegenis, J., Georgakakis, D., Angelidaki, I., Mavris, V., 2007. Optimization of biogas production by co-digesting whey with diluted poultry manure. Appl. Energy 32 (13), 2147–2160. He, X., Yin, J., Liu, J., Chen, T., Shen, D., 2019. Characteristics of acidogenic fermentation for volatile fatty acid production from food waste at high concentrations of NaCl. Bioresour. Technol. 271, 244–250. Iacovidou, E., Ohandja, D.G., Voulvoulis, N., 2012. Food waste co-digestion with sewage sludge – realising its potential in the UK. J. Environ. Manage. 112 (24), 267–274. Jankowska, E., Chwialkowska, J., Stodolny, M., Oleskowicz-Popiel, P., 2017. Volatile fatty acids production during mixed culture fermentation – The impact of substrate complexity and pH. Chem. Eng. J. 326, 901–910. Jiang, J., Zhang, Y., Li, K., Wang, Q., Gong, C., Li, M., 2013. Volatile fatty acids production from food waste: effects of pH, temperature, and organic loading rate. Bioresour. Technol. 143, 525–530. Jiang, J., Gong, C., Wang, J., Tian, S., Zhang, Y., 2014. Effects of ultrasound pre-treatment on the amount of dissolved organic matter extracted from food waste. Bioresour. Technol. 155 (2), 266–271. Jin, Y., Gao, M., Li, H., Lin, Y., Wang, Q., Tu, M., Ma, H., 2019. Impact of nanoscale zerovalent iron on volatile fatty acid production from food waste: key enzymes and microbial community. J. Chem. Technol. Biotechnol. https://doi.org/10.1002/jctb. 6127. Kim, K.-H., Eom, I.-Y., Lee, S.-M., Cho, S.-T., Choi, I.-G., Choi, J.W., 2010. Applicability of sub- and supercritical water hydrolysis of woody biomass to produce monomeric sugars for cellulosic bioethanol fermentation. J. Ind. Eng. Chem. 16 (6), 918–922. Lee, W.S., Chua, A.S.M., Yeoh, H.K., Ngoh, G.C., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83–99. Li, H., Ma, H., Liu, T., et al., 2019a. An excellent alternative composite modifier for cathode catalysts prepared from bacterial cellulose doped with Cu and P and its utilization in microbial fuel cell. Bioresour. Technol. 289, 121661. Li, Y.Y., Zhang, X., Xu, H., Mu, H., Hua, D., Jin, F., Meng, G., 2019b. Acidogenic properties of carbohydrate-rich wasted potato and microbial community analysis: Effect of pH. J. Biosci. Bioeng. 128 (1), 50–55. Liu, H., Hang, X., Bo, Y., Zu, Y., He, L., Bo, F., Ma, H., 2016. Enhanced volatile fatty acid production by a modified biological pretreatment in anaerobic fermentation of waste activated sludge. Chem. Eng. J. 284, 194–201. Ma, Y., Liu, Y., 2019. Turning food waste to energy and resources towards a great environmental and economic sustainability: An innovative integrated biological approach. Biotechnol. Adv. Ma, H., Yang, J., Jia, Y., Wang, Q., Tashiro, Y., Sonomoto, K., 2016. Stillage reflux in food waste ethanol fermentation and its by-product accumulation. Bioresour. Technol. 209, 254–258. Min, K.S., Khan, A.R., Kwon, M.K., Jung, Y.J., Yun, Z., Kiso, Y., 2005. Acidogenic fermentation of blended food-waste in combination with primary sludge for the production of volatile fatty acids. J. Chem. Technol. Biotechnol. Biotechnol. 80 (8), 909–915. Nzeteu, C.O., Trego, A.C., Abram, F., O'Flaherty, V., 2018. Reproducible, high-yielding, biological caproate production from food waste using a single-phase anaerobic reactor system. Biotechnol. Biofuels 11 (1), 108–122. Pham, T.P.T., Kaushik, R., Parshetti, G.K., Mahmood, R., Balasubramanian, R., 2015. Food waste-to-energy conversion technologies: current status and future directions. Waste Manage. 38, 399–408. Ren, N., Guo, W., Liu, B., Cao, G., Jie, D., 2011. Biological hydrogen production by dark fermentation: challenges and prospects towards scaled-up production. Curr. Opin. Biotechnol. 22 (3), 365–370. Ren, Y., Yu, M., Wu, C., Wang, Q., Gao, M., Huang, Q., Liu, Y., 2018. A comprehensive review on food waste anaerobic digestion: Research updates and tendencies. Bioresour. Technol. 247, 1069–1076. Roghair, M., Liu, Y.C., Strik, D.P.B.T.B., Weusthuis, R.A., Bruins, M.E., Buisman, C.J.N., 2018. Development of an effective chain elongation process from acidified food waste and ethanol into n-caproate. Front. Bioeng. Biotechnol. 6, 1–11. Rotstein, O.D., Pruett, T.L., Fiegel, V.D., Nelson, R.D., Simmons, R.L., 1985. Succinic acid, a metabolic by-product of Bacteroides species, inhibits polymorphonuclear leukocyte function. Infect. Immun. 48 (2), 402–408. Suo, Y., Ren, M., Yang, X., Liao, Z., Fu, H., Wang, J., 2018. Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production with high butyrate/ acetate ratio. Appl. Microbiol. Biotechnol. 102 (10), 4511–4522. Tang, J.L., Wang, X.C., Hu, Y.S., Zhang, Y.M., Li, Y.Y., 2016. Effect of pH on lactic acid production from acidogenic fermentation of food waste with different types of inocula. Bioresour. Technol. 224, 544–552. Wang, Y., Guo, W.-Q., Xing, D.-F., Chang, J.-S., Ren, N.-Q., 2014b. Hydrogen production using biocathode single-chamber microbial electrolysis cells fed by molasses wastewater at low temperature. Int. J. Hydrogen Energy 39 (33), 19369–19375. Wang, K., Yin, J., Shen, D., Na, L., 2014a. Anaerobic digestion of food waste for volatile fatty acids (VFAs) production with different types of inoculum: Effect of pH. Bioresour. Technol. 161 (6), 395–401. Warnecke, T., Gill, R.T., 2005. Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Microb. Cell Fact. 4 (1), 25–32. Wilhelm, H.H., Brian, J.B.W., 2014. The genus Vagococcus. Lactic Acid Bacteria: Biodiversity and Taxonomy. John Wiley & Sons, Ltd., pp. 229–237. Wu, Q., Guo, W., Zheng, H., Luo, H., Feng, X., Yin, R., Ren, N., 2016. Enhancement of volatile fatty acid production by co-fermentation of food waste and excess sludge without pH control: the mechanism and microbial community analyses. Bioresour. Technol. 216, 653–660.
4. Conclusion Residue from saccharification and centrifugation of food waste ethanol fermentation (RSCFW) showed the highest concentration of VFAs (267.8 mg COD/g VS) at pH 6.5, and the butyric acid became dominant product with pH controlled at 5.5 and 6.5. The dominant bacteria analyzed by microbial analysis showed the change from bacteria for acetic acid production to butyric production. Acknowledgements This research was supported by the Open Research Fund Program of Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry (CP-2019-YB7), Beijing Municipal Science and Technology Project (Z181100002418016). The authors also acknowledged the support by Fundamental Research Funds for the Central Universities (TW2019014). References APHA, 2012. Standard methods for the examination of water and wastewater. American Public Health. Association, Washington, DC. Bengtsson, S., Hallquist, J., Werker, A., Welander, T., 2008. Acidogenic fermentation of industrial wastewaters: effects of chemostat retention time and pH on volatile fatty acids production. Biochem. Eng. J. 40 (3), 492–499. Chen, Y., Luo, J., Yan, Y., Feng, L., 2013b. Enhanced production of short-chain fatty acid by co-fermentation of waste activated sludge and kitchen waste under alkaline conditions and its application to microbial fuel cells. Appl. Energy 102 (2), 1197–1204. Chen, H., Meng, H., Nie, Z., Zhang, M., 2013a. Polyhydroxyalkanoate production from fermented volatile fatty acids: effect of pH and feeding regimes. Bioresour. Technol. 128, 533–538. Dareioti, M.A., Kornaros, M., 2014. Effect of hydraulic retention time (HRT) on the anaerobic co-digestion of agro-industrial wastes in a two-stage CSTR system. Bioresour. Technol. 167 (3), 407–415. Dong-Hoon, K., Sang-Hyoun, K., Kyung-Won, J., Mi-Sun, K., Hang-Sik, S., 2011. Effect of initial pH independent of operational pH on hydrogen fermentation of food waste. Bioresour. Technol. 102 (18), 8646–8652. Feng, W., Wei-Ying, L., Xue-Nong, Y., 2015. Two-phase anaerobic co-digestion of food waste and sewage sludge. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 71 (1), 52–58. Fusco, V., Quero, G.M., Cho, G.-S., Kabisch, J., Meske, D., Neve, H., Bockelmann, W., Franz, C.M.A.P., 2015. The genus Weissella: taxonomy, ecology and biotechnological potential. Front. Microbiol. 6, 155.
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Water Res. 42 (18), 4637–4644. Yu, M., Wu, C., Wang, Q., Sun, X., Ren, Y., Li, Y.-Y., 2018. Ethanol prefermentation of food waste in sequencing batch methane fermentation for improved buffering capacity and microbial community analysis. Bioresour. Technol. 248, 187–193. Yuan, H., Chen, Y., Zhang, H., Jiang, S., Zhou, Q., Gu, G., 2006. Improved bioproduction of short-chain fatty acids (SCFAs) from excess sludge under alkaline conditions. Environ. Sci. Technol. 40 (6), 2025. Zhang, P., Chen, Y., Zhou, Q., 2009. Waste activated sludge hydrolysis and short-chain fatty acids accumulation under mesophilic and thermophilic conditions: effect of pH. Water Res. 43 (15), 3735–3742. Zhou, M., Yan, B., Wong, J.W.C., Zhang, Y., 2018. Enhanced volatile fatty acids production from anaerobic fermentation of food waste: a mini-review focusing on acidogenic metabolic pathways. Bioresour. Technol. 248, 68–78.
Yan, Y., Feng, L., Zhang, C., Wisniewski, C., Zhou, Q., 2010. Ultrasonic enhancement of waste activated sludge hydrolysis and volatile fatty acids accumulation at pH 10.0. Water Res. 44 (11), 3329–3336. Yan, L., Liu, S., Liu, Q., et al., 2019. Improved performance of simultaneous nitrification and denitrification via nitrite in an oxygen-limited SBR by alternating the DO. Bioresour. Technol. 275, 153–162. Yang, X., Wan, C., Lee, D.J., Du, M., Pan, X., Wan, F., 2014. Continuous volatile fatty acid production from waste activated sludge hydrolyzed at pH 12. Bioresour. Technol. 168, 173–179. Yin, J., Yu, X., Zhang, Y., Shen, D., Wang, M., Long, Y., Chen, T., 2016. Enhancement of acidogenic fermentation for volatile fatty acid production from food waste: effect of redox potential and inoculum. Bioresour. Technol. 216, 996–1003. Yu, G.-H., He, P.-J., Shao, L.-M., He, P.-P., 2008. Toward understanding the mechanism of improving the production of volatile fatty acids from activated sludge at pH 10.0.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Volatile fatty acids production from saccharification residue from food waste ethanol fermentation: Effect of pH and microbial community
T
Yong Jina,b, Yujia Lina,b, Pan Wangc, Runwen Jina,b, Ming Gaoa,b, Qunhui Wanga,b, ⁎ Tien-Chin Changd, Hongzhi Maa,b, a
Department of Environmental Engineering, University of Science and Technology Beijing, 100083, China Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, Beijing 100083, China c Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry, Beijing Technology and Business University, Beijing 100048, China d Institute of Environmental Engineering and Management, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Rd., Taipei 106, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Food waste Anaerobic fermentation VFAs pH Community analysis
In this study, residue from saccharification and centrifugation of food waste ethanol fermentation was used as substrate to produce volatile fatty acids. The effects of different pH (5.5, 6.5, and uncontrolled) on the VFAs concentration, composition, acidogenic efficiency and microbial community distribution were investigated. The results showed that the highest concentration of VFAs was 267.8 ± 8.9 mg COD/g VS at pH of 6.5, and the highest percentage of butyric acid (79.8%) was followed by propionic acid and acetic acid at the end of the reaction. Microbial analysis showed that the contents of Vagococcus and Actinomyces increased, while the contents of Bacteroides and Fermentimonas decreased during anaerobic fermentation. The comparative high pH induced the accumulation of butyric acid. This study provides a new idea for the step anaerobic fermentation of food waste to produce alcohol and acid simultaneously.
1. Introduction Food waste, with high water content, high organic matter, high salt content and perishability, has drawn many researches’ interest on its resource utilization (Ren et al., 2018). Compared with general treatments, such as incineration, landfill, composting, the most effective way is anaerobic fermentation, which produces acid or methane (Zhou et al., 2018). However, the Volatile Fatty Acids (VFAs) production has a shorter time than methane fermentation (Ma and Liu, 2019). VFAs produced by the fermentation of food waste can be used as a carbon source for nitrogen and phosphorus removal in wastewater treatment (Wang et al., 2014b), as a raw material for producing biodegradable plastics (Jiang et al., 2014), and vinyl acetate (Jiang et al., 2013). In addition, VFAs can be further used by microorganisms to produce hydrogen (Chen et al., 2013b), methane and biodiesel (Pham et al., 2015), PHA and electricity (Chen et al., 2013a), and for chain elongation (Roghair et al., 2018), etc. Although VFAs production can achieve energy recovery of food waste, the separate fermentation of food waste under high load may cause problems such as excessive Na+ concentration, excessive accumulation of organic acids, and rapid pH decrease due to excessive
⁎
acidification rate (Gelegenis et al., 2007). Besides food waste, sludge also has great value for energy recovery (Li et al., 2019a). But it is easy to be alkaline by hydrolysis alone, resulting in excessive pH, high ammonia nitrogen concentration, high heavy metal content and pathogen inhibition, which may adversely affect the fermentation process (Iacovidou et al., 2012). Therefore, some researchers have proposed the co-fermented of food waste and sludge to produce VFAs, which could not only make efficient use of organic substances in food waste and sludge, but also reduce the pollution (Chen et al., 2013b; Feng et al., 2015; Wu et al., 2016). During anaerobic fermentation, the fermented products and their composition are affected by various factors. As an important environmental factor, pH can not only affect the activity of microbial enzymes, but also determine the presence of VFAs (Garcia-Aguirre et al., 2017). Many researchers studied the effect of pH on the hydrolysis and acidification of organic solid waste by dynamically adjusting the pH value. Some researchers believed that anaerobic fermentation under alkaline conditions could inhibit the activity of methanogens, which was conducive to the accumulation of VFAs (Yan et al., 2010; Yang et al., 2014; Yu et al., 2008; Zhang et al., 2009). However, in the practical application of fermentation engineering, anaerobic fermentation will
Corresponding author. E-mail address: [email protected] (H. Ma).
https://doi.org/10.1016/j.biortech.2019.121957 Received 1 July 2019; Received in revised form 2 August 2019; Accepted 3 August 2019 Available online 07 August 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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gradually reduce the pH to acidic. If it is adjusted to alkaline conditions, Na+ will accumulate and inhibit the activity of acidogenic microorganisms, resulting in a decrease in subsequent acid production capacity. In addition, it would not be economically feasible to adjust the pH to alkaline conditions (Jankowska et al., 2017). Therefore, it is more economical and reasonable to control pH to optimize acid production under weakly acidic conditions. Jiang et al tested the effect of anaerobic digestion of food waste at pH 5, 6, 7 and without pH control under medium temperature conditions (35 °C) (Jiang et al., 2013). The results showed that when the pH was 6, the acidogenic efficiency of food waste was the best. Wang and Yin et al. used aerobic and anaerobic activated sludge as inoculum to study the effect of pH (4, 5, 6) on the yield of VFAs using food waste as a substrate (Wang et al., 2014a). The results showed that pH 4 was more favorable for the hydrolysis of organic matter than other pH values, and the best pH for VFAs production was at pH 6. However, there were few researches on microbial community changes at different stages under faintly acid conditions. The organic matter in the food waste was rich and complicated. In order to further utilize the various organic substances in a high-efficiency way, researchers used the stillage of food waste to produce ethanol or lactic acid, and the residue was used for composting or sanitary landfill (Ma et al., 2016). However, this method cannot fully utilize the organic substances in food waste, the remaining saccharification residue still contained a small amount of sugar and protein, and there existed little research on how to treat the residue. The purpose of this study is to further utilize the residue from saccharification and centrifugation of food waste ethanol fermentation (RSCFW). Co-fermentation was carried out by adjusting different pH and inoculating anaerobic sludge with acclimation to investigate the effect of pH on VFAs production, composition and microbial community structure during anaerobic fermentation.
Table 1 Characteristics of RSCFW and Seeding sludge.
2. Materials and methods
stirring was controlled by a programmable logic controller (PLC) at 250 r/min, and the pH was adjusted with 3 mol/L HCl solution and 3 mol/L NaOH solution. The values were 5.5 and 6.5, respectively, and the anaerobic environment was maintained during the whole fermentation process. The fermentation time was 72 h and samples were obtained every 12 h.
Parameters
RSCFW
Seeding sludge
pH TS/% VS/% C (%) N (%) Carbohydrate (%) Protein (%)
6.3 ± 0.1 26.3 ± 2.3 25.1 ± 2.1 46.1 ± 3.2 3.6 ± 0.2 48.9 ± 4.4 15.9 ± 1.6
6.5 ± 0.1 8.4 ± 0.4 5.0 ± 0.1 28.73 ± 2.3 3.9 ± 0.2 64.2 ± 5.9 11.2 ± 1.1
Note: the pH in RSCFW was measured after adding deionized water by 2:1.
Fig. 1. Variation of VFAs and ethanol concentration at different pH value.
2.1. Materials 2.1.1. Residue from saccharification and centrifugation of food waste (RSCFW) The food waste was taken from a canteen in a university in Beijing. After preliminary sorting, crushing and passing through a 3-mm sieve, it was diluted with water by a mass ratio of 2:1 (food waste: water) and placed in a water bath. Saccharification enzyme with the dose of 100 U/ g was added, the saccharification was carried with rotation 60 r/min at 60 °C in water bath for 6 h, and then centrifuged at 12,000 r/min for 10 min, the liquid and residue of food waste were detained (Ma et al., 2016).
2.3. Analytic methods All of the samples were centrifuged for 10 min at 12,000 r/min and then the supernatants were filtered through a 0.45 µm membrane. All the tests were carried out in parallel triplicates and the average values were determined. 2.3.1. VFAs and ethanol test VFAs and ethanol were measured by gas chromatograph (GC-2010 plus, Shimadzu, Japan) equipped with a capillary column (DB-FFAP) and a flame ionization detection (FID). The temperature rise procedure was as follows: initial temperature of 60 °C for 2 min, increased to 100 °C at 20 °C/min, held for 1 min, increased to 170 °C at 10 °C/min, maintained for 1 min, and raised to 210 °C at 20 °C/min for 2 min. The inlet (SO1 PL1) temperature, FID temperature, and injection volume were 220 °C, 240 °C, and 0.4 µL, respectively (Yu et al., 2018).
2.1.2. Seeding sludge with acclimation The sludge was taken from a domestic sewage treatment plant in Beijing, first concentrated at ambient temperature for 24 h, and the precipitated sludge was treated in a water bath at 105 °C for 2 h in order to kill methanogens. Then it was transferred to a 500-mL shake flask and added to the food waste (5% g VS/g) for anaerobic acclimate for two weeks. When the pH went down from 7.8 to about 6.5 (Liu et al., 2016), the anaerobic sludge was centrifuged at 4000 r/min for 10 min to obtain acclimation sludge. The basic properties of saccharified residue and acclimation sludge were shown in Table 1.
2.3.2. Other analytical methods Soluble Chemical Oxygen Demand (SCOD) was measured using standard methods (APHA, 2012). TS was calculated from the dry organic waste from the filter after heating in an oven at 105 °C, and VS was measured after the dry organic waste was burned in a muffle furnace. The pH was measured by a pH probe (pHSJ-3F, China). The acidification yield (ηa ) (Eq. (1)) was calculated as the ratio of the cumulative VFAs and final concentration of SCOD in the leachate
2.2. Experimental setup The RSCFW and seeding sludge were mixed at a mass ratio of 2.5:1 (Volatile Solid (VS) ratio) and Total Solid (TS) of 10%, then put into a reaction apparatus, and then was added 2-bromoethanesulfonic sodium salt (BES, Adamas, ≥98%) (50 mmol/L) (Jin et al., 2019). The temperature was controlled by the heating device at (37 ± 1) °C, the 2
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(Jankowska et al., 2017):
ηa =
VFAs × 100% SCOD
(1)
The VFAs/SCOD indicated how many soluble substances were converted into VFAs by fermentation. The higher the ratio, the higher the VFAs conversion rate is. 2.4. Microbial community analysis 2.4.1. DNA extraction and PCR amplification Microbial DNA was extracted from V4 to V5 samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to manufacturer’s protocols (Yan et al., 2019). The V4-V5 region of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min) using primers 515F ( 5′-GTGCCAGCMGCCGCGG-3′), 907R (5′-CCGTCAATTCMTTTRAGTTT-3′), where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate of 20-μL mixtures containing 4 μL of 5× FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA, for amplification. Finally, the PCR products were sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). 2.4.2. Illumina MiSeq sequencing Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions and quantified using QuantiFluor™-ST (Promega, USA). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA556916). 2.4.3. Processing of sequencing data Raw fastq files were demultiplexed, quality-filtered using QIIME with the following criteria: (1) 300 bp reads were truncated at any site receiving an average quality score < 20 over a 10 bp sliding window, discarding the truncated reads that were shorter than 50 bp; (2) no ambiguous bases were allowed, and no mismatches were allowed in the primer sequence. Operational units (OTUs) were clustered with 97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/ ), and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the Silva (SSU115) 16S rRNA database using confidence threshold of 70%. The general differences of microbial community structure were evaluated by principal component analysis (PCA) in Canoco 5.0 (Microcomputer Power, USA) using the relative abundances of the whole microbial communities. 3. Results and discussion 3.1. Effect of pH on the VFAs concentration and composition (1) VFAs and ethanol concentration In the process of hydrolysis and acidification, microorganisms secrete extracellular hydrolase, which hydrolyzes complex macromolecular organic substances (proteins, polysaccharides, oils, etc.) into small molecular organic substances (such as monosaccharides, amino acids, glycerol and long-chain fatty acids). Acidogenic microorganisms used these small molecules to produce pyruvic acid, lactic acid, etc., which were then converted to VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) or other products (Yin et al., 2016). The results of acid production of saccharified residue
Fig. 2. Variation of VFAs composition at different pH value.
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Table 2 VFAs composition under different pH value at the end of fermentation (%). pH
Acetic acid
Propionic acid
Isobutyric acid
Butyric acid
Isovaleric acid
Valeric acid
5.5 6.5 Uncontrolled
6.9 4.6 20.7
7.7 9.4 12.7
1.1 1.5 1.9
81.0 79.3 61.4
2.0 3.5 3.3
1.3 1.7 0
uncontrolled pH, respectively. Jiang et al found that when pH was 6, the concentration of VFAs reached a maximum of 37.60 g/L, 9 times of those without pH control (Jiang et al., 2013). In addition, Wang et al. found that pH control at 5.0 to 6.5 favored hydrolysis and acidification, and the highest VFAs yield (0.918 g/g VSSremoval) was obtained at pH 6 (Wang et al., 2014a). At the end of the fermentation, the concentrations of VFAs were 232.5 ± 11.3, 232.4 ± 11.2, and 98.7 ± 9.3 mg COD/g VS, respectively. Ren et al. (2011) observed that acid-producing bacteria were inhibited at pH < 4.0, and methanogens were activated when pH > 6.5 (Yuan et al., 2006). It could be seen from Fig. 1 that when the pH was controlled, the initial VFAs concentration was different, indicated that the initial pH might affect the dissolution of the substrate (Yang et al., 2014). At the beginning of the reaction (0–12 h), the concentration of VFAs decreased rapidly, indicated that the initial pH change would affect the activity of acid-producing bacteria. Then VFAs increased slowly, indicating that the acid-producing bacteria adapted to the pH environment. At the end of fermentation, the concentration of VFAs at pH 5.5 still increased, indicating that the pH condition was suitable for the growth of acid-producing bacteria (Nzeteu et al., 2018). In addition to VFAs, ethanol was also produced, albeit at a lower level. There were many studies on the production of ethanol by anaerobic fermentation of food waste. However, the substrate in this study was RSCFW, which produced low ethanol concentration by co-fermentation with sludge. When the pH was 6.5, the concentration of ethanol reached the highest (27.9 ± 0.7 mg/g VS). There might be the following reasons, the main component of RSCFW was protein (nitrogen source), but ethanol fermentation mainly used sugar, so ethanol production was very low.
Fig. 3. Variation of SCOD and VFAs/SCOD at different pH value. Table 3 Samples information for bacterial community structure analysis. Name
Sample source
Sampling time (h)
1_solid 2_mixed 3_fermented
RSCFW RSCFW & seeding sludge Output in the reactor at pH 6.5
0 0 72
(2) VFAs composition According to the composition of products, anaerobic fermentation could be divided into different fermentation types, such as butyric acid fermentation, propionic acid fermentation, mixed acid fermentation, and so on. By investigating anaerobic digestion of food waste under different acidic conditions for VFAs production, there were different results (Lee et al., 2014). Min et al. (2005) found that the maximum propionate fraction (80%) was observed at pH 6.5 in a continuous reactor. Bengtsson et al. (2008) reported that acetic and propionic acids were the main components during the acidogenic fermentation of different paper mill wastewaters at pH 6.0. Dong-Hoon et al. (2011) found that the production of butyrate was highest at pH 8.0 comparing with other pH at 5.0, 6.0, 7.0 and 9.0. Jiang et al. (2013) found that when the pH was at 5.0 or uncontrolled, acetate was the dominant product, followed by butyrate, propionate, and valerate; and when the pH was at 6.0 and 7.0, butyrate became the main product. Wang et al. (2014a) found that butyrate was the primary product under pH 5.0 with the percentage of butyrate above 80%. The difference among these studies is mainly due to the different choices of substrates and reactors. In our study, we explored the composition of the product under pH control with RSCFW as the substrate (Fig. 2). As shown in Fig. 2, the products mainly contained acetic acid, propionic acid, and butyric acid under different pH conditions. When the pH was 5.5 and 6.5, the acetic acid ratio reached the highest at the 12th hour, followed by propionic and butyric acids. At the end of fermentation, the order of these three main products were converse. When
Fig. 4. The bacterial community analysis from different sample source at phylum level.
were shown in Fig. 1. It could be seen that the concentration of VFAs without pH control was the lowest in 0–36 h. Those with pH of 5.5 decreased continuously, while those with pH of 6.5 decreased first and then rose rapidly. The highest concentration of 267.8 mg COD/g VS was obtained with pH of 6.5 at the 36 h, 3 and 10 times higher than those pH of 5.5 and 4
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Table 4 Phylogenetic classification of the 16S rRNA gene sequences. Phylum
Class (%)
Solid
Mixed
Fermented
Genus (%)
Solid
Mixed
Fermented
Firmicutes
Bacilli
98.58
7.01
35.92
Clostridia
0.0021
32.16
33.59
Weissella Bacteroides Clostridium_sensu_stricto_1 Vagococcus Bacillus Fermentimonas Actinomyces Terrisporobacter Turicibacter Romboutsia
59 0 0.0021 0.011 36 0 0 0 0 0
0.028 40 21 5.5 0.19 12 1.8 3.6 3.7 1.7
0.011 12 23 34 0.57 5.2 9.1 3.6 3.4 1.6
Globicatella norank_f_Rikenellaceae Norank_o_Chloroplast
0 0 1.4
1.2 1.9 0
0.93 0.0081 0
Bacteroidetes
Erysipelotrichia
0
3.74
3.45
Bacteroidia
0
54.36
17.50
Actinobacteria
Actinobacteria
0
2.02
9.37
Ruminococcaceae_NK4A214_group
0
0.20
1.0
Cyanobacteria
Oxyphotobacteria
1.36
0
0
Leuconostoc
1.1
0
0
Others
Others
0.06
0.71
0.16
Others
3.0
7.8
5.5
It could be seen that when the pH was 6.5, the concentration of SCOD decreased rapidly in the first 12 h and then increased slowly. As for pH 5.5 and uncontrolled, the concentration of SCOD tended to be stable. At the end of fermentation, the concentration of SCOD with uncontrolled was highest (492.75 ± 6.5 mg/g VS). The result showed that the solubility of SCOD was higher with pH uncontrolled, which was contrary to (Jiang et al., 2013). It was probably due to the rapid acidification which resulted in the death of most of the bacteria in the inoculum and the dissolution of the organic matter in the cell, resulting in the higher concentration of SCOD (Warnecke and Gill, 2005). On the other hand, when the pH was 6.5, VFAs/SCOD was significantly higher than the other two groups, and the highest VFAs conversion rate reached to 61.6 ± 2.5% at 36 h, less than those without pH control (83.46%) (Wu et al., 2016), this was because the organic matter in our substrate was much less. At the end of fermentation, the VFAs/SCOD at pH 5.5 was higher than pH 6.5, it indicated that the strains with pH 5.5 might adapt to the acid environment (Tang et al., 2016). And this can also explain the phenomenon of the continuous increase of VFAs at pH 5.5 in Fig. 1.
Fig. 5. The bacterial community analysis from different sample source at genus level.
3.3. Microbial community analysis
the pH was uncontrolled, although the butyric acid ratio continued to increase, the acetic acid content was higher than that of the other two groups. Moreover, Table 2 showed the VFAs composition under different pH at the end of fermentation. It can be seen that acetic acid fermentation was the main reaction at the first 24 h (pH 5.5–36 h), and then butyric acid production gradually increased with the change of dominant strain. Propionic acid concentration decreased slightly and the overall trend was not obvious. At the end of fermentation, the ratio of butyric acid was 81.0%, 79.3%, and 61.5% under different pH conditions. The results showed that under the pH 5.5–6.5, the dominant strain was for butyric acid production. This may be due to the chain elongation under pH control, i.e. the acetic acid of two carbons elongated to butyric acid of four carbons (Suo et al., 2018).
During the anaerobic fermentation, the microbial activity would be affected by different treatment conditions. He et al. (2019) studied the changes of microbial community in the production of VFAs from food waste by high concentration of NaCl. Wu et al. (2016) investigated the effect of uncontrolled pH on microbial community in the co-fermentation of food waste and excess sludge to produce VFAs. Li et al. (2019b) studied the variation of microbial community at pH 6, 7, and 8. However, few studies have concentrated on the changes of microbial community structure at pH 5.5–6.5. Therefore, it was necessary to further analyze the variation of substrate, inoculum and fermented products under pH control. Sample information of bacterial community structure in this study was shown in Table 3. As shown in the Fig. 4 about the phylum level analysis, all the dominant bacteria were Firmicutes, accounting for 98.6%, 42.9%, and 73.0%, respectively. Firmicutes were the dominant hydrolyzed bacteria which were capable of degrading sugars, fats, and proteins for acid production (Dareioti and Kornaros, 2014). In the mixed stage, another dominant group was Bacteroidetes (54.4%), and finally it decreased to 17.5% in the fermented stage. Bacteroidetes was a major bacterium for methane production, and it was inhibited with the BES addition (Yu et al., 2018). The Actinobacteria, increased slightly from 2.0% to 9.4%, could promote the hydrolysis of cellulose and the decomposition of glucose (Kim et al., 2010). The increase in the abundance of
3.2. Effect of pH on the acidogenic efficiency In the acidogenic fermentation, insoluble substances in RSCFW were gradually hydrolyzed into soluble proteins, fats, etc. And they would further be fermented into VFAs, H2, and CO2 with the microorganisms, thus, the SCOD of RSCFW would be changed. Besides, it was necessary to study the acidogenic efficiency by calculating the VFAs/SCOD. The variation of SCOD and VFAs/SCOD at different pH are shown in the Fig. 3. 5
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Actinobacteria also indicated the decomposition of organic matter in the pH 6.5 could better promote the acid production process. In order to further demonstrate the functional evolution of the bacterial microbial community, the bacteria were analyzed at the genus level, and the composition and comparison of major species were shown in Table 4 and Fig. 5, respectively. It could be seen from Fig. 5 that the major seven genera were Weissella, Bacteroides, Clostridium_sensu_stricto_1, Vagococcus, Bacillus, Fermentimonas, and Actinomyces, they belonged to the class Bacilli and Clostridia (Table 4). In the RSCFW, the dominant genus Weissella (59%) belonged to the Firmicutes, producing CO2 from carbohydrate metabolism with lactic acid and acetic acid as major end products from sugar metabolism (Fusco et al., 2015). In the mixed stage, the dominant genus changed from Weissella to Bacteroides (40%), which was a type of anaerobe within the Bacteroidetes phylum, and succinic acid was the major fatty acid byproduct of Bacteroides metabolism (Rotstein et al., 1985). In the fermented stage, the major genus was the Vagococcus, which was mainly producing lactic acid as the end product (Wilhelm and Brian, 2014). Compared the change of dominant bacteria between the mixed and fermented stage, it could be found that the Bacteroides and the Fermentimonas decreased from 40% to 12% and 12% to 5.2%, respectively. Conversely, the Vagococcus and the Actinomyces were increased from 5.5% to 34% and 1.8% to 9.1%, respectively. With the pH 6.5, more lactic acid was produced in the first stage, and then it would be degraded, so there was more butyric acid produced from acetic acid, and there would be a chain elongation in the next stage.
Garcia-Aguirre, J., Aymerich, E., González-Mtnez de Goñi, J., Esteban-Gutiérrez, M., 2017. Selective VFA production potential from organic waste streams: assessing temperature and pH influence. Bioresour. Technol. 244, 1081–1088. Gelegenis, J., Georgakakis, D., Angelidaki, I., Mavris, V., 2007. Optimization of biogas production by co-digesting whey with diluted poultry manure. Appl. Energy 32 (13), 2147–2160. He, X., Yin, J., Liu, J., Chen, T., Shen, D., 2019. Characteristics of acidogenic fermentation for volatile fatty acid production from food waste at high concentrations of NaCl. Bioresour. Technol. 271, 244–250. Iacovidou, E., Ohandja, D.G., Voulvoulis, N., 2012. Food waste co-digestion with sewage sludge – realising its potential in the UK. J. Environ. Manage. 112 (24), 267–274. Jankowska, E., Chwialkowska, J., Stodolny, M., Oleskowicz-Popiel, P., 2017. Volatile fatty acids production during mixed culture fermentation – The impact of substrate complexity and pH. Chem. Eng. J. 326, 901–910. Jiang, J., Zhang, Y., Li, K., Wang, Q., Gong, C., Li, M., 2013. Volatile fatty acids production from food waste: effects of pH, temperature, and organic loading rate. Bioresour. Technol. 143, 525–530. Jiang, J., Gong, C., Wang, J., Tian, S., Zhang, Y., 2014. Effects of ultrasound pre-treatment on the amount of dissolved organic matter extracted from food waste. Bioresour. Technol. 155 (2), 266–271. Jin, Y., Gao, M., Li, H., Lin, Y., Wang, Q., Tu, M., Ma, H., 2019. Impact of nanoscale zerovalent iron on volatile fatty acid production from food waste: key enzymes and microbial community. J. Chem. Technol. Biotechnol. https://doi.org/10.1002/jctb. 6127. Kim, K.-H., Eom, I.-Y., Lee, S.-M., Cho, S.-T., Choi, I.-G., Choi, J.W., 2010. Applicability of sub- and supercritical water hydrolysis of woody biomass to produce monomeric sugars for cellulosic bioethanol fermentation. J. Ind. Eng. Chem. 16 (6), 918–922. Lee, W.S., Chua, A.S.M., Yeoh, H.K., Ngoh, G.C., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83–99. Li, H., Ma, H., Liu, T., et al., 2019a. An excellent alternative composite modifier for cathode catalysts prepared from bacterial cellulose doped with Cu and P and its utilization in microbial fuel cell. Bioresour. Technol. 289, 121661. Li, Y.Y., Zhang, X., Xu, H., Mu, H., Hua, D., Jin, F., Meng, G., 2019b. Acidogenic properties of carbohydrate-rich wasted potato and microbial community analysis: Effect of pH. J. Biosci. Bioeng. 128 (1), 50–55. Liu, H., Hang, X., Bo, Y., Zu, Y., He, L., Bo, F., Ma, H., 2016. Enhanced volatile fatty acid production by a modified biological pretreatment in anaerobic fermentation of waste activated sludge. Chem. Eng. J. 284, 194–201. Ma, Y., Liu, Y., 2019. Turning food waste to energy and resources towards a great environmental and economic sustainability: An innovative integrated biological approach. Biotechnol. Adv. Ma, H., Yang, J., Jia, Y., Wang, Q., Tashiro, Y., Sonomoto, K., 2016. Stillage reflux in food waste ethanol fermentation and its by-product accumulation. Bioresour. Technol. 209, 254–258. Min, K.S., Khan, A.R., Kwon, M.K., Jung, Y.J., Yun, Z., Kiso, Y., 2005. Acidogenic fermentation of blended food-waste in combination with primary sludge for the production of volatile fatty acids. J. Chem. Technol. Biotechnol. Biotechnol. 80 (8), 909–915. Nzeteu, C.O., Trego, A.C., Abram, F., O'Flaherty, V., 2018. Reproducible, high-yielding, biological caproate production from food waste using a single-phase anaerobic reactor system. Biotechnol. Biofuels 11 (1), 108–122. Pham, T.P.T., Kaushik, R., Parshetti, G.K., Mahmood, R., Balasubramanian, R., 2015. Food waste-to-energy conversion technologies: current status and future directions. Waste Manage. 38, 399–408. Ren, N., Guo, W., Liu, B., Cao, G., Jie, D., 2011. Biological hydrogen production by dark fermentation: challenges and prospects towards scaled-up production. Curr. Opin. Biotechnol. 22 (3), 365–370. Ren, Y., Yu, M., Wu, C., Wang, Q., Gao, M., Huang, Q., Liu, Y., 2018. A comprehensive review on food waste anaerobic digestion: Research updates and tendencies. Bioresour. Technol. 247, 1069–1076. Roghair, M., Liu, Y.C., Strik, D.P.B.T.B., Weusthuis, R.A., Bruins, M.E., Buisman, C.J.N., 2018. Development of an effective chain elongation process from acidified food waste and ethanol into n-caproate. Front. Bioeng. Biotechnol. 6, 1–11. Rotstein, O.D., Pruett, T.L., Fiegel, V.D., Nelson, R.D., Simmons, R.L., 1985. Succinic acid, a metabolic by-product of Bacteroides species, inhibits polymorphonuclear leukocyte function. Infect. Immun. 48 (2), 402–408. Suo, Y., Ren, M., Yang, X., Liao, Z., Fu, H., Wang, J., 2018. Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production with high butyrate/ acetate ratio. Appl. Microbiol. Biotechnol. 102 (10), 4511–4522. Tang, J.L., Wang, X.C., Hu, Y.S., Zhang, Y.M., Li, Y.Y., 2016. Effect of pH on lactic acid production from acidogenic fermentation of food waste with different types of inocula. Bioresour. Technol. 224, 544–552. Wang, Y., Guo, W.-Q., Xing, D.-F., Chang, J.-S., Ren, N.-Q., 2014b. Hydrogen production using biocathode single-chamber microbial electrolysis cells fed by molasses wastewater at low temperature. Int. J. Hydrogen Energy 39 (33), 19369–19375. Wang, K., Yin, J., Shen, D., Na, L., 2014a. Anaerobic digestion of food waste for volatile fatty acids (VFAs) production with different types of inoculum: Effect of pH. Bioresour. Technol. 161 (6), 395–401. Warnecke, T., Gill, R.T., 2005. Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Microb. Cell Fact. 4 (1), 25–32. Wilhelm, H.H., Brian, J.B.W., 2014. The genus Vagococcus. Lactic Acid Bacteria: Biodiversity and Taxonomy. John Wiley & Sons, Ltd., pp. 229–237. Wu, Q., Guo, W., Zheng, H., Luo, H., Feng, X., Yin, R., Ren, N., 2016. Enhancement of volatile fatty acid production by co-fermentation of food waste and excess sludge without pH control: the mechanism and microbial community analyses. Bioresour. Technol. 216, 653–660.
4. Conclusion Residue from saccharification and centrifugation of food waste ethanol fermentation (RSCFW) showed the highest concentration of VFAs (267.8 mg COD/g VS) at pH 6.5, and the butyric acid became dominant product with pH controlled at 5.5 and 6.5. The dominant bacteria analyzed by microbial analysis showed the change from bacteria for acetic acid production to butyric production. Acknowledgements This research was supported by the Open Research Fund Program of Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry (CP-2019-YB7), Beijing Municipal Science and Technology Project (Z181100002418016). The authors also acknowledged the support by Fundamental Research Funds for the Central Universities (TW2019014). References APHA, 2012. Standard methods for the examination of water and wastewater. American Public Health. Association, Washington, DC. Bengtsson, S., Hallquist, J., Werker, A., Welander, T., 2008. Acidogenic fermentation of industrial wastewaters: effects of chemostat retention time and pH on volatile fatty acids production. Biochem. Eng. J. 40 (3), 492–499. Chen, Y., Luo, J., Yan, Y., Feng, L., 2013b. Enhanced production of short-chain fatty acid by co-fermentation of waste activated sludge and kitchen waste under alkaline conditions and its application to microbial fuel cells. Appl. Energy 102 (2), 1197–1204. Chen, H., Meng, H., Nie, Z., Zhang, M., 2013a. Polyhydroxyalkanoate production from fermented volatile fatty acids: effect of pH and feeding regimes. Bioresour. Technol. 128, 533–538. Dareioti, M.A., Kornaros, M., 2014. Effect of hydraulic retention time (HRT) on the anaerobic co-digestion of agro-industrial wastes in a two-stage CSTR system. Bioresour. Technol. 167 (3), 407–415. Dong-Hoon, K., Sang-Hyoun, K., Kyung-Won, J., Mi-Sun, K., Hang-Sik, S., 2011. Effect of initial pH independent of operational pH on hydrogen fermentation of food waste. Bioresour. Technol. 102 (18), 8646–8652. Feng, W., Wei-Ying, L., Xue-Nong, Y., 2015. Two-phase anaerobic co-digestion of food waste and sewage sludge. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 71 (1), 52–58. Fusco, V., Quero, G.M., Cho, G.-S., Kabisch, J., Meske, D., Neve, H., Bockelmann, W., Franz, C.M.A.P., 2015. The genus Weissella: taxonomy, ecology and biotechnological potential. Front. Microbiol. 6, 155.
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Bioresource Technology 292 (2019) 121957
Y. Jin, et al.
Water Res. 42 (18), 4637–4644. Yu, M., Wu, C., Wang, Q., Sun, X., Ren, Y., Li, Y.-Y., 2018. Ethanol prefermentation of food waste in sequencing batch methane fermentation for improved buffering capacity and microbial community analysis. Bioresour. Technol. 248, 187–193. Yuan, H., Chen, Y., Zhang, H., Jiang, S., Zhou, Q., Gu, G., 2006. Improved bioproduction of short-chain fatty acids (SCFAs) from excess sludge under alkaline conditions. Environ. Sci. Technol. 40 (6), 2025. Zhang, P., Chen, Y., Zhou, Q., 2009. Waste activated sludge hydrolysis and short-chain fatty acids accumulation under mesophilic and thermophilic conditions: effect of pH. Water Res. 43 (15), 3735–3742. Zhou, M., Yan, B., Wong, J.W.C., Zhang, Y., 2018. Enhanced volatile fatty acids production from anaerobic fermentation of food waste: a mini-review focusing on acidogenic metabolic pathways. Bioresour. Technol. 248, 68–78.
Yan, Y., Feng, L., Zhang, C., Wisniewski, C., Zhou, Q., 2010. Ultrasonic enhancement of waste activated sludge hydrolysis and volatile fatty acids accumulation at pH 10.0. Water Res. 44 (11), 3329–3336. Yan, L., Liu, S., Liu, Q., et al., 2019. Improved performance of simultaneous nitrification and denitrification via nitrite in an oxygen-limited SBR by alternating the DO. Bioresour. Technol. 275, 153–162. Yang, X., Wan, C., Lee, D.J., Du, M., Pan, X., Wan, F., 2014. Continuous volatile fatty acid production from waste activated sludge hydrolyzed at pH 12. Bioresour. Technol. 168, 173–179. Yin, J., Yu, X., Zhang, Y., Shen, D., Wang, M., Long, Y., Chen, T., 2016. Enhancement of acidogenic fermentation for volatile fatty acid production from food waste: effect of redox potential and inoculum. Bioresour. Technol. 216, 996–1003. Yu, G.-H., He, P.-J., Shao, L.-M., He, P.-P., 2008. Toward understanding the mechanism of improving the production of volatile fatty acids from activated sludge at pH 10.0.
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