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Three-ways changed in headspace air on anaerobic fermentation
Bioresource Technology 289 (2019) 121684
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Three-ways changed in headspace air on anaerobic fermentation a
a
Leigang Li , Yu Li , Reham Yasser Farouk a b
a,b
, Yuanyuan Wang
T
a
College of Engineering, Huazhong Agricultural University, Wuhan 430070, PR China Department of Agricultural Engineering, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt
A R T I C LE I N FO
A B S T R A C T
Keywords: Headspace air Dark fermentation Partial hydrogen pressure Homoacetogenesis
Different headspace condition has a great influence on fermentation process. In this study, whey protein was used as substrate, and the headspace air was changed in three different ways (H2 mixed N2, H2 mixed CO2, N2 with different sparging rates) to explore the effects of these three methods on products. The result showed that H2 mixed with CO2 is more conducive to acid production. Homoacetogenesis played a central role in fermentation process. There is a turning point in the role of hydrogen and Homoacetogenesis, which is when the partial pressure of hydrogen is 0.268 atm. In the first two conditions, the acid concentration increased with the increase of hydrogen percentage. Nitrogen sparging way is adverse to acid production, but conducive to gas production.
1. Introduction As a kind of biodegradation, anaerobic fermentation conforms to the natural law and has no obvious side effects. Nowadays, in the case of the energy crisis, dark fermentation is getting more and more attention due to the presence of hydrogen as a product. The concept of deriving energy and commodities by processing organic material is defined as a biorefinery (Yang et al., 2015). Biological hydrogen production by dark fermentation may assume a central role in the use of organic waste in a biorefinery concept (Alibardi et al., 2014). Biohydrogen production and its advancement are important aspects of current biorefinery techniques that focus on high value-added chemicals for pharmaceutical, cosmetics nutritional, and biofuel industries (Yang et al., 2011; Sostaric et al., 2012). Not only that, but also the value of the volatile fatty acids (VFAs) produced in the process is potential. The representing biodegradable materials consist of a series of substances belonging to the three main categories of organic matter: carbohydrates, proteins and lipids. A series of value-added bioproducts can be produced from these organics by biological processes, including bioenergy, chemicals, biodiesel and bioplastics (Xia et al., 2013; Lee et al., 2014; Pham et al., 2015; Alibardi and Cossu, 2015). Protein is a potential raw material with a lower conversion rate of 40–70%, while glucose can reach 88% by anaerobic fermentation (Shen et al., 2017). Some studies demonstrated that the hydrolysis of protein was very slow because protein was unsusceptible to protease cleavage in its native folded conformation (Herman et al., 2006; Yin et al., 2016). Yin et al (2016) added trypsin or alkaline protease in residual postfermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%,
respectively. The dark fermentation process is influenced by numerous parameters, including organic loading rate, hydraulic detention time, temperature, pH, etc. These factors have a significant impact on the result generated by organic waste fermentation, leading to large differences in the data reported in the scientific literature (see Fig. 1). Although some current studies have explored protein fermentation, the mechanism analysis and the choice of experimental factors are far from enough. Bai et al. (2004) focused on the effects of protein on hydrogen yields in batch conditions from mixtures of glucose or starch, as a source of carbohydrate, and peptone as a source of protein. Shen et al. (2017) found tofu could directly produce VFAs through the Stickland reaction, while egg white was converted to lactate and VFAs simultaneously. About 30–40% of total protein remained in all groups after fermentation. The mainly fermentation pathway of amino acids is coupling degradation through the Stickland reaction. Under mixedculture, mixed-amino acid conditions, uncoupled degradation of amino acids only occur if there is a shortage of amino acids that can act as electron acceptors. Piotr et al. (2017) cites a considerable part of the stoichiometric equations of pure amino acid degradation. But this reaction accounts for a quite small proportion of the fermentation process. Ramsay and Pullammanappallil (2001) had listed all stoichiometric numbers for amino acid fermentation (catabolic reactions only). Liu et al. (2012) had given the total reaction equation of VFA accumulation according to the compositions and concentrations of amino acids from the protein biodecomposition of sewage sludge in the reactors. And it described as (β depend on the stoichiometry coefficient):
E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.biortech.2019.121684 Received 28 May 2019; Received in revised form 16 June 2019; Accepted 18 June 2019 Available online 19 June 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 289 (2019) 121684
L. Li, et al.
Fig. 1. Metabolic pathway of the protein degradation.
0.1 g/L CaCl2·2H2O, 0.1 g/L MnCl2·6H2O, 0.05 g/L ZnCl2, 0.1 g/L Na2MoO4, 0.05 g/L MgSO4, and 0.01 g/L NiCl2·6H2O. The medium was added every 48 h, and the enrichment was completed after 5 times (Liu and Wang, 2016). Finally, the concentration of volatile solids (VS) and total solids (TS) of seed sludge were 11.37% and 22.05%, respectively. In the process of the experiment, whey protein comes from MYPROTEIN in the UK. It contains 82% whey protein, 4% sugar and 12.5% fat.
CN0.29 H2.05 O0.74 + α H2 O→ βHAc CH3 COOH + βHPr CH3 CH2 COOH + βIBu CH3 CH2 CH2 COOH + βHBu CH3 CH2 CH2 COOH + βIVa CH3 CH2 CH2 CH2 COOH + βHVa CH3 CH2 CH2 CH2 COOH
(1)
Hydrogen is produced during the decomposition of amino acids, but quickly consumed due to its decomposition characteristics. So there is no hydrogen in the reaction equation. Most researchers think that hydrogen partial pressure (PH2 ) is an important factor affecting the results, mainly because in many biochemical reactions, the transfer of H+ and electrons can make the reaction proceed normally. Moreover, the reduction of PH2 reduces the Gibbs free energy of those reactions with hydrogen as the product makes the reaction more conducive to hydrogen generation, such as homoacetogenesis reaction, which is detrimental to hydrogen consumption when the PH2 is lowered. It is worth noting that the role of other gases cannot be easily ignored. Headspace of fermenter, which affect hydrogen pressure directly, has been listed as an important factor affecting fermentation in recent years. A considerable number of scholars have studied the effects of headspace pressure or partial pressure on the fermentation process (Lee et al., 2012; Yan et al., 2014; Valero et al., 2016; Sarkar et al., 2017; Zhou et al., 2017; Shen et al., 2017; Hua Yan et al., 2017). However, little research has been done on the use of protein as raw material, and there is a lack of more detailed description of the role of partial pressure of H2 and CO2. To investigate the effect of headspace gas on protein dark fermentation process, we used 82% protein-containing whey protein liquid as fermentation material. A semi continuous fermentation method was adopted to explore the effects of three methods of changing the headspace on the fermentation process.
2.2. Fermentation process setup and operation The test adopts a semi-continuous fermentation method in which the headspace gas in the fermentation reactor is changed in three ways: hydrogen and carbon dioxide mixture (Group 1), hydrogen and nitrogen mixture (Group 2) and nitrogen (Group 3). The experimental device is shown in Fig. 2. The test period at each level was 10.5 days. A 500 ml serum bottle (working volume is 400 ml), used as fermenter, is connected to an air pump and a 5 L air bag (gas volume is 3 L). The reactors were initially inoculated at a rate of 37.5% (v/v). The
2. Materials and methods 2.1. Inoculum and fermentation medium Seed sludge was a mixture of fresh pig manure and biogas slurry. The sludge was filtered and thermally pretreated at 120 °C for 30 min to inactivate methanogen and pH was adjusted to 5.0 (approx.). The medium used for fermentation contained 10 g/L glucose, 5 g/L peptone and sufficient amounts of inorganic supplements, including 4 g/L NaCl, 2 g/L (NH4)2SO4, 1.5 g/L KH2PO4, 0.2 g/L CoCl2·6H2O, 0.2 g/L FeSO4,
Fig. 2. Schematic diagram of fermentation system. 2
Bioresource Technology 289 (2019) 121684
L. Li, et al.
fermentation bottle is placed in a 37 ± 0.5 °C water bath to maintain the temperature. After inoculation, the gas is kept for 30 s to replace headspace. Then, to maintain the anaerobic environment, the fermenter is sealed with the butyl rubber stopper air. In Group 1 and 2, the pump runs for 3 min every 2 h to mix the headspace air; In Group 3, the gas is bubbled continuously at different rates (0 L/d, 20 L/d, 40 L/d, 60 L/d). In the experiment, the protein concentration in the fermentation substrate was 6.25 g/L and the pH was changed to about 6.00. The hydraulic detention time (HRT) was 36 h. Headspace gas composition was shown in Table 1. The substrate and air bags were replaced every 36 h, and the obtained liquid and gas sample were measured.
Except for 0% H2 level, the variation on hydrogen volume of other levels was lower than that in Group 1. There were some biochemical reactions consume hydrogen, including homoacetogenesis, HVa reaction, Propanol reaction, etc. Reductive deamination from sole amino acid is favorable and is hydrogen consuming (Dong et al., 2009). Since the only difference between the setting factors of Groups 1 and 2 was carbon dioxide and nitrogen in headspace of fermenter, it is inferred that the main cause of this phenomenon is the homoacetogenesis, which produced HAc with carbon dioxide and hydrogen as raw materials. It could be seen from the gas composition of Group 3 that the sparging of N2 was favorable for the formation of carbon dioxide; as the sparging rate increased, the volume of carbon dioxide changes were 94 ml, 144 ml, 214 ml and 295 ml, respectively, and the carbon dioxide content was 2.98% rises to 8.83%, indicating that the gas sparging rate had a great influence on the formation of carbon dioxide. The variations of hydrogen volume were 70 ml, 93 ml, 68 ml and 46 ml, respectively. This means that hydrogen production can be promoted at a nitrogen rate of 20 L/d. The blowing of nitrogen can cause a certain agitation and promote the escape of gas. But too fast flow is not conducive to the stability of the strain.
2.3. Analytical methods
3.2. VFAs concentration
Solluted Chemical oxygen demand (SCOD), VS and TS were determined according to standard methods (APHA, 1998). The soluble protein was quantified by the Bradford method using bovine serum albumin as the standard (Noaman and Coorssen, 2018). Volumetric biogas composition, including CH4, CO2, N2, and H2 was analyzed using a gas chromatograph (FULI; GC9790II; China) equipped with a thermal conductivity detector and packed column (Porapak Q column [3 m × 3 mm] cascading with a TDX-01 column [2 m × 3 mm]). The carrier gas was helium. The injector, oven, and detector temperatures were 150, 200 and 120 °C, respectively. Volumetric hydrogen and methane yields were calculated by multiplying the total yield by the corresponding volume percentages. The VFAs, including acetic acid (HAc), propionic acid (HPr), isobutyric acid (IBu), butyric acid (HBu), Isovaleric acid (IVa) and valeric acid (HVa) were measured using the same gas chromatograph equipped with a flame ionized detector (FID) and column detector (Kromat, KB-Wax; 30 m × 0.32 mm × 0.33 mm). The carrier gas was nitrogen at a flow rate of 75 ml/min. The inlet and detector temperatures were both 250 °C. The oven temperature was originally 80 °C and was increased to 200 °C at a rate of 20 °C/min.
The VFAs concentration in the fermentation vessel was obtained by recording the average value of the stable period, as shown in Fig. 3. In Group 1, the graph revealed that the VFAs concentration fluctuated all through. In 0% H2 level, the VFAs concentration was the lowest (5873.972 mg/L) among the five levels. Then it rose in 25% H2 level and dropped in 50% H2 level. The peak concentration (6554.295 mg/L) of VFAs was occurred at 100% H2 level. The trends for the concentration of HAc, IVa, IBu and HBu were similar, and this trend was similar to VFAs concentration. HPr and HVa did not change much in different conditions. According to Daltons law of partial pressures, the final PH2 were 0.018 atm, 0.203 atm, 0.441 atm, 0.645 atm and 0.793 atm, respectively. Many opinions believed that low PH2 favored the formation of acids (Zhou et al., 2017; Sarkar et al., 2017). But the observed phenomena were somewhat different from this view. Therefore, we had to consider the effects of homoacetogenesis and carbon dioxide partial pressure (PCO2 ). The PCO2 mainly acted on the homoacetogenesis, but we cannot estimate its other effects. Kim et al. (2006) reported that high PCO2 had little effect on H2-producing bacteria but inhibitory effect on other microorganisms such as acetogens and lactic acid bacteria which were competitive with H2-producing bacteria. The mechanism for this effect was not clearly stated. Eq. (2) is the reaction involved in acetate production without H2 generation, which are supported by the existence of acetate-producing bacteria which could consume H2 and CO2.
Table 1 Headspace composition.
1 2 3 4 5
Group 1
Group 2
Group 3 (N2)
0%H2 + 100%CO2 25%H2 + 75%CO2 50%H2 + 50%CO2 75%H2 + 25%CO2 100%H2 + 0%CO2
0%H2 + 100%N2 25%H2 + 75%N2 50%H2 + 50%N2 75%H2 + 25%N2 100%H2 + 0%N2
0 L/d 20 L/d 40 L/d 60 L/d –
3. Results and discussion 3.1. Analysis of headspace gas
4H2 + 2CO2 → CH3 COOH + 2H2 O( T= 37°C, ΔG′ = −74.54 kJ)
Table 2 showed the volume and composition of the headspace, all data taken from the mean value of the stable period. The volume of the headspace in Group 1 and 2 decreased slightly, but did not exceed 8%, while a small amount of gases was produced in Group 3. In Group 1, the percentage of hydrogen changed little except 100% H2 level. From 0% to 100% H2 level, the variations on hydrogen volume were 60 ml, −114 ml, −105 ml, −198 ml and −389 ml, respectively. The hydrogen gas volume decreases except for 0% H2 level, and with the increase of hydrogen percentage, the reduction of hydrogen also increased. The change of carbon dioxide is contrary to that of hydrogen, the variations on carbon dioxide volume were −288 ml, −162 ml, −122 ml, −11 ml and 284 ml, respectively. A similar situation occurred in Group 2. The small change in the percentage of gas content is in line with expectations, as this avoided the impact of excessive changes in gas composition on the test process. But in Group 2, the volume of remaining gas is slightly higher than Group 1. From 0% to 100% H2 level, the variations of hydrogen volume were 86 ml, −68 ml, −94 ml, −129 ml and −348 ml, respectively.
(2)
Δr Gm (T ) = △r Gmθ (T ) + RTlnQ Q=
[c (CH3 COOH )/ c θ] [PH2/ pθ ]4 [PCO2/ pθ ]2
Under standard conditions, the Gibbs free energy of the reaction is negative, which means that the reaction is easier to occur in the fermentation. △r Gmθ (T ) and Q reflected the influence of temperature and pressure on the reaction, respectively. As the fermentation proceeded, when the PH2 and PCO2 were reduced to a low enough level or the HAc concentration was high enough, the reaction will reach equilibrium, and the effect of PH2 is greater than PCO2 . As H2 level increased from 0% to 50%, the balance between PH2 and homoacetogenesis occurred in this range. To better determine when this balance occurred, it should be analyzed in conjunction with the results of Group 2. Between 0% and 50% H2 levels in Group 2, the concentration of HAc fell below 25% H2 level and began to make a recovery. In Fig. 4, through linear fitting, it 3
Bioresource Technology 289 (2019) 121684
L. Li, et al.
Table 2 The biogas volume and composition. Component
Volume (ml)
Group 1 (%)
Volume (ml)
Group 2 (%)
Volume (ml)
Group 3 (%)
1
H2 N2 CH4 CO2
2823
2.11 1.83 0 96.06
2974
2.90 94.57 0 1.53
3165
2.22 94.80 0 2.98
2
H2 N2 CH4 CO2
2759
23.05 1.28 0 75.67
2824
24.16 71.38 0 4.46
3237
2.88 92.68 0 4.44
3
H2 N2 CH4 CO2
2788
50.05 0.52 0 49.43
2892
48.60 90.28 0 1.62
3282
2.07 91.41 0 6.52
4
H2 N2 CH4 CO2
2804
73.19 0.46 0 26.35
2872
73.84 22.86 0 3.30
3341
1.37 89.80 0 8.83
5
H2 N2 CH4 CO2
2901
90.01 0.18 0 9.81
2944
90.07 2.28 0 7.65
The deviation of test results is no more than 5%.
was found that the equilibrium point is between 25% and 50% H2 levels, and the PH2 was about 0.268 atm. Therefore, we could conclude that the promotion of low PH2 had been offset when the PH2 was greater than 0.268 atm. The fermentation medium can be easily supersaturated with H2 due to liquid-to-gas mass transfer limitation (Ljunggren et al., 2011; Zhang et al., 2012), this may be one of the reasons why PH2 promotes acid production at extremely low pressure. In Group 2, the trend of VFAs concentration declined below 25% H2 level and then rises. The maximum VFAs concentration was found in 100% H2 level (6331.405 mg/L); and the minimum concentration was found in 25% H2 level (5849.187 mg/L), which was different from Group 1. The concentration changes of HAc and HBu were basically the same as that of VFAs. There was no significant difference in IBu, HPr, IVa and HVa concentration. Compared with Group 1, the main difference was HAc. In Fig. 4, we found that in the same H2 level, the HAc concentration of Group 1 was 2–14% higher than that in Group 2. With the increase in PH2 and the decrease of PCO2 , the concentration of HAc became higher. And in 25% H2 level, CO2 had the greatest enhancement effect. As the PH2 continued to rise, the HAc concentration increased, but the difference in HAc concentration of the two groups becomes smaller, which indicated that the influence of PH2 on homoacetogenesis was greater. The PH2 in each level was 0.026 atm, 0.213 atm, 0.428 atm, 0.650 atm and 0.793 atm, respectively, which was similar to Group 1. So the main reason for the difference of acid concentration between Group 1 and 2 came from CO2. In Group 1, the PCO2 were 0.846 atm,
Fig. 4. Concentration of acetate in Group 1 and 2 with its fitting curve.
0.666 atm, 0.435 atm, 0.232 atm and 0.086 atm, respectively. But in Group 2, the PCO2 was very low, ranging from 0.013 to 0.067 atm. The PCO2 will directly affect the effect of homoacetogenesis and change the metabolic distribution. High PCO2 could significantly increase HAc concentration.
Fig. 3. VFA concentration in the reactor (Group 1: H2 + CO2, Group 2: H2 + N2, Group 3: N2, the deviation of test results is no more than 5%). 4
Bioresource Technology 289 (2019) 121684
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In Group 3, it is observable that the VFAs concentration of Group 3 is significantly lower than that of Group 1 and 2. The main sources of this gap were acetate and butyrate. Besides, the concentration of IVa was higher, which may be the reason for the low concentration of acetate and butyrate. With the increase in flow rate, the concentration of VFAs fluctuated all through, and kept the maximum when the flow rate was 0 L/d, reaching 5501.806 mg/L. The main difference between the four levels was HBu. For example, at 0 L/d, the concentration of HBu was 1688.053 mg/L, and at 60 L/d, it was 1192.852 mg/L, which decreased by 29.38%. As the sparging rate increased, the trends of HPr, IBu, HBu, IVa and HVa concentration were the same, rising during 20–40 L/d but falling during 0–20 L/d and 40–60 L/d. HAc concentration was first decreased and then rises; no more than that, there is little difference between 0 L/d and 60 L/d (about 1870 mg/L), and between 20 L/d and 40 L/d (about 1700 mg/L). Different from other level, there is no artificial mixing process in the headspace air of 0 L/d, which may be one of the reasons for the change of acid production pathway. Gas sparging can cause sludge floating, gas escaping, etc. In brief, changes in the headspace can affect the concentration and distribution of VFAs, and even the metabolic pathways of microorganisms.
69–77. Alibardi, L., Muntoni, A., Polettini, A., 2014. Hydrogen and waste: illusions, challenges and perspectives. Waste Manage. 34, 2425–2426. APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association/American Water Works Association/Water Environment Federation, Washington, DC, USA. Bai, M., Cheng, S., Chao, Y., 2004. Effects of substrate components on hydrogen fermentation of multiple substrates. Water Sci. Technol. 50, 209–216. Dong, L., Zhenhong, Y., Yongming, S., Xiaoying, K., Yu, Z., 2009. Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation. Int. J. Hydrogen Energy 34, 812–820. Herman, R., Gao, Y., Storer, N.P., 2006. Acid-induced unfolding kinetics in simulated gastric digestion of proteins. Regulatory Toxicol. Pharmacol. Rtp 46 (1), 93–99. Hua Yan, B., Selvam, A., Wong, J.W.C., 2017. Influence of acidogenic headspace pressure on methane production under schematic of diversion of acidogenic off-gas to methanogenic reactor. Bioresour. Technol. 245, 1000–1007. Kim, D.H., Han, S.K., Kim, S.H., Shin, H.S., 2006. Effect of gas sparging on continuous fermentative hydrogen production. Int. J. Hydrogen Energy 31 (15), 2158–2169. Lee, K.S., Tseng, T.S., Liu, Y.W., Hsiao, Y.D., 2012. Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy. Int. J. Hydrogen Energy 37 (20), 15556–15562. 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 (1), 83–99. Liu, Y., Wang, Y., 2016. Directional enhancement of fermentative coproduction of hydrogen and acetic acid from glucose via control of headspace pressure. Int. J. Hydrogen Energy 42 (7). Liu, H., Wang, J., Liu, X., Fu, B., Chen, J., Yu, H.Q., 2012. Acidogenic fermentation of proteinaceous sewage sludge: effect of ph. Water Res. 46 (3). Ljunggren, M., Willquist, K., Zacchi, G., Van Niel, Ed W.J., 2011. A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus. Biotechnol. Biofuels 4 (1), 31. Noaman, N., Coorssen, J.R., 2018. Coomassie does it (better): a Robin Hood approach to total protein quantification. Anal. Biochem. 556, 53–56. Pham, T.P.T., Kaushik, R., Parshetti, G.K., 2015. Food waste-to-energy conversion technologies current status and future directions. Waste Manage. 38, 399–408. Piotr, S., Krzysztof, P., Jan, C., Jolanta, B., 2017. Alternative utilization of protein-rich waste by its conversion into biogas in co-fermentation conditions. Pol. J. Environ. Stud. 26 (3), 1225–1231. Ramsay, I.R., Pullammanappallil, P.C., 2001. Protein degradation during anaerobic wastewater treatment: derivation of stoichiometry. Biodegradation 12 (4), 247–256. Sarkar, O., Butti, S.K., Venkata Mohan, S., 2017. Acidogenesis driven by hydrogen partial pressure towards bioethanol production through fatty acids reduction. Energy 118, 425–434. Shen, D.S., Yin, J., Yu, X.Q., Wang, M.Z., Long, Y.Y., Shentu, J.L., Chen, T., 2017. Acidogenic fermentation characteristics of different types of protein-rich substrate food waste to produce volatile fatty acids. Bioresour. Technol. 227, 125–132. Sostaric, M., Klinar, D., Bricelj, M., Golob, J., Berovic, M., Likozar, B., 2012. Growth, lipid extraction and thermal degradation of the microalga Chlorella vulgaris. New Biotechnol. 29, 325–331. Valero, D., Montes, Jesús A., Rico, José Luis, Rico, C., 2016. Influence of headspace pressure on methane production in biochemical methane potential (BMP) tests. Waste Manage. 48 (1), 193–198. Xia, A., Cheng, J., Lin, R., Liu, J., Zhou, J., Cen, K., 2013. Sequential generation of hydrogen and methane from glutamic acid through combined photo-fermentation and methanogenesis. Bioresour. Technol. 131, 146–151. Yan, B.H., Selvam, A., Xu, S.Y., Wong, J.W.C., 2014. A novel way to utilize hydrogen and carbon dioxide in acidogenic reactor through homoacetogenesis. Bioresour. Technol. 159, 249–257. Yang, X., Choi, H.S., Park, C., Kim, S.W., 2015. Current states and prospects of organic waste utilization for biorefineries. Renew. Sustain. Energy Rev. 49, 335–349. Yang, Z., Guo, R., Xu, X., Fan, X., Luo, S., 2011. Hydrogen and methane production from lipid-extracted microalgal biomass residues. Int. J. Hydrogen Energy 36, 3465–3470. Yin, B., Liu, H.B., Wang, Y.Y., Bai, J., Liu, H., Fu, B., 2016. Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: protease catalysis of refractory protein. Bioresour. Technol. 203, 124–131. Zhang, F., Zhang, Y., Chen, M., Zeng, R.J., 2012. Hydrogen supersaturation in thermophilic mixed culture fermentation. Int. J. Hydrogen Energy 37 (23), 17809–17816. Zhou, M., Zhou, J., Tan, M., Du, J., Yan, B., Wong, J.W.C., Zhang, Y., 2017. Enhanced carboxylic acids production by decreasing hydrogen partial pressure during acidogenic fermentation of glucose. Bioresour. Technol. 245, 44–51.
4. Conclusions The different headspace air has a significant impact on the fermentation process. PH2 , PCO2 and homoacetogenesis have a greater impact on the distribution of acid production. When the PH2 and PCO2 are high, the homoacetogenesis will be promoted, which causing an increase in acid concentration. But the effect of PH2 is greater than that of PCO2 . Homoacetogenesis play a role part when the PH2 was greater than 0.268 atm. Addition of CO2 is beneficial to HAc production, the PCO2 will directly affect the effect of homoacetogenesis and change the metabolic distribution. When the PCO2 is 0.666 atm, the promotion effect reaches the maximum. High PCO2 could significantly increase HAc concentration. In Group 3, the concentrations of acids are both lower than Group 1 and 2. Sparging way is not conducive to acid formation, but conducive to gas production. Accordingly, if we aim to produce acid, the fermentation system can be divided into two phases of hydrolysis acid production and acetic acid production, and the two-phase acid production is promoted when the gas for hydrolyzing acid is introduced into the second phase. Acknowledgements This work was financially supported by grants from National Natural Science Foundation of China (no. 51606083), Fundamental Research Funds for the Central Universities of China (no. 2662008JC006), and Youth scholar promotion project of Dabei agricultural fund (2017DBN007). References Alibardi, L., Cossu, R., 2015. Effects of carbohydrate, protein and lipid content of organic waste on hydrogen production and fermentation products. Waste Manage. 47 (Pt A),
5
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Three-ways changed in headspace air on anaerobic fermentation a
a
Leigang Li , Yu Li , Reham Yasser Farouk a b
a,b
, Yuanyuan Wang
T
a
College of Engineering, Huazhong Agricultural University, Wuhan 430070, PR China Department of Agricultural Engineering, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt
A R T I C LE I N FO
A B S T R A C T
Keywords: Headspace air Dark fermentation Partial hydrogen pressure Homoacetogenesis
Different headspace condition has a great influence on fermentation process. In this study, whey protein was used as substrate, and the headspace air was changed in three different ways (H2 mixed N2, H2 mixed CO2, N2 with different sparging rates) to explore the effects of these three methods on products. The result showed that H2 mixed with CO2 is more conducive to acid production. Homoacetogenesis played a central role in fermentation process. There is a turning point in the role of hydrogen and Homoacetogenesis, which is when the partial pressure of hydrogen is 0.268 atm. In the first two conditions, the acid concentration increased with the increase of hydrogen percentage. Nitrogen sparging way is adverse to acid production, but conducive to gas production.
1. Introduction As a kind of biodegradation, anaerobic fermentation conforms to the natural law and has no obvious side effects. Nowadays, in the case of the energy crisis, dark fermentation is getting more and more attention due to the presence of hydrogen as a product. The concept of deriving energy and commodities by processing organic material is defined as a biorefinery (Yang et al., 2015). Biological hydrogen production by dark fermentation may assume a central role in the use of organic waste in a biorefinery concept (Alibardi et al., 2014). Biohydrogen production and its advancement are important aspects of current biorefinery techniques that focus on high value-added chemicals for pharmaceutical, cosmetics nutritional, and biofuel industries (Yang et al., 2011; Sostaric et al., 2012). Not only that, but also the value of the volatile fatty acids (VFAs) produced in the process is potential. The representing biodegradable materials consist of a series of substances belonging to the three main categories of organic matter: carbohydrates, proteins and lipids. A series of value-added bioproducts can be produced from these organics by biological processes, including bioenergy, chemicals, biodiesel and bioplastics (Xia et al., 2013; Lee et al., 2014; Pham et al., 2015; Alibardi and Cossu, 2015). Protein is a potential raw material with a lower conversion rate of 40–70%, while glucose can reach 88% by anaerobic fermentation (Shen et al., 2017). Some studies demonstrated that the hydrolysis of protein was very slow because protein was unsusceptible to protease cleavage in its native folded conformation (Herman et al., 2006; Yin et al., 2016). Yin et al (2016) added trypsin or alkaline protease in residual postfermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%,
respectively. The dark fermentation process is influenced by numerous parameters, including organic loading rate, hydraulic detention time, temperature, pH, etc. These factors have a significant impact on the result generated by organic waste fermentation, leading to large differences in the data reported in the scientific literature (see Fig. 1). Although some current studies have explored protein fermentation, the mechanism analysis and the choice of experimental factors are far from enough. Bai et al. (2004) focused on the effects of protein on hydrogen yields in batch conditions from mixtures of glucose or starch, as a source of carbohydrate, and peptone as a source of protein. Shen et al. (2017) found tofu could directly produce VFAs through the Stickland reaction, while egg white was converted to lactate and VFAs simultaneously. About 30–40% of total protein remained in all groups after fermentation. The mainly fermentation pathway of amino acids is coupling degradation through the Stickland reaction. Under mixedculture, mixed-amino acid conditions, uncoupled degradation of amino acids only occur if there is a shortage of amino acids that can act as electron acceptors. Piotr et al. (2017) cites a considerable part of the stoichiometric equations of pure amino acid degradation. But this reaction accounts for a quite small proportion of the fermentation process. Ramsay and Pullammanappallil (2001) had listed all stoichiometric numbers for amino acid fermentation (catabolic reactions only). Liu et al. (2012) had given the total reaction equation of VFA accumulation according to the compositions and concentrations of amino acids from the protein biodecomposition of sewage sludge in the reactors. And it described as (β depend on the stoichiometry coefficient):
E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.biortech.2019.121684 Received 28 May 2019; Received in revised form 16 June 2019; Accepted 18 June 2019 Available online 19 June 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Metabolic pathway of the protein degradation.
0.1 g/L CaCl2·2H2O, 0.1 g/L MnCl2·6H2O, 0.05 g/L ZnCl2, 0.1 g/L Na2MoO4, 0.05 g/L MgSO4, and 0.01 g/L NiCl2·6H2O. The medium was added every 48 h, and the enrichment was completed after 5 times (Liu and Wang, 2016). Finally, the concentration of volatile solids (VS) and total solids (TS) of seed sludge were 11.37% and 22.05%, respectively. In the process of the experiment, whey protein comes from MYPROTEIN in the UK. It contains 82% whey protein, 4% sugar and 12.5% fat.
CN0.29 H2.05 O0.74 + α H2 O→ βHAc CH3 COOH + βHPr CH3 CH2 COOH + βIBu CH3 CH2 CH2 COOH + βHBu CH3 CH2 CH2 COOH + βIVa CH3 CH2 CH2 CH2 COOH + βHVa CH3 CH2 CH2 CH2 COOH
(1)
Hydrogen is produced during the decomposition of amino acids, but quickly consumed due to its decomposition characteristics. So there is no hydrogen in the reaction equation. Most researchers think that hydrogen partial pressure (PH2 ) is an important factor affecting the results, mainly because in many biochemical reactions, the transfer of H+ and electrons can make the reaction proceed normally. Moreover, the reduction of PH2 reduces the Gibbs free energy of those reactions with hydrogen as the product makes the reaction more conducive to hydrogen generation, such as homoacetogenesis reaction, which is detrimental to hydrogen consumption when the PH2 is lowered. It is worth noting that the role of other gases cannot be easily ignored. Headspace of fermenter, which affect hydrogen pressure directly, has been listed as an important factor affecting fermentation in recent years. A considerable number of scholars have studied the effects of headspace pressure or partial pressure on the fermentation process (Lee et al., 2012; Yan et al., 2014; Valero et al., 2016; Sarkar et al., 2017; Zhou et al., 2017; Shen et al., 2017; Hua Yan et al., 2017). However, little research has been done on the use of protein as raw material, and there is a lack of more detailed description of the role of partial pressure of H2 and CO2. To investigate the effect of headspace gas on protein dark fermentation process, we used 82% protein-containing whey protein liquid as fermentation material. A semi continuous fermentation method was adopted to explore the effects of three methods of changing the headspace on the fermentation process.
2.2. Fermentation process setup and operation The test adopts a semi-continuous fermentation method in which the headspace gas in the fermentation reactor is changed in three ways: hydrogen and carbon dioxide mixture (Group 1), hydrogen and nitrogen mixture (Group 2) and nitrogen (Group 3). The experimental device is shown in Fig. 2. The test period at each level was 10.5 days. A 500 ml serum bottle (working volume is 400 ml), used as fermenter, is connected to an air pump and a 5 L air bag (gas volume is 3 L). The reactors were initially inoculated at a rate of 37.5% (v/v). The
2. Materials and methods 2.1. Inoculum and fermentation medium Seed sludge was a mixture of fresh pig manure and biogas slurry. The sludge was filtered and thermally pretreated at 120 °C for 30 min to inactivate methanogen and pH was adjusted to 5.0 (approx.). The medium used for fermentation contained 10 g/L glucose, 5 g/L peptone and sufficient amounts of inorganic supplements, including 4 g/L NaCl, 2 g/L (NH4)2SO4, 1.5 g/L KH2PO4, 0.2 g/L CoCl2·6H2O, 0.2 g/L FeSO4,
Fig. 2. Schematic diagram of fermentation system. 2
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fermentation bottle is placed in a 37 ± 0.5 °C water bath to maintain the temperature. After inoculation, the gas is kept for 30 s to replace headspace. Then, to maintain the anaerobic environment, the fermenter is sealed with the butyl rubber stopper air. In Group 1 and 2, the pump runs for 3 min every 2 h to mix the headspace air; In Group 3, the gas is bubbled continuously at different rates (0 L/d, 20 L/d, 40 L/d, 60 L/d). In the experiment, the protein concentration in the fermentation substrate was 6.25 g/L and the pH was changed to about 6.00. The hydraulic detention time (HRT) was 36 h. Headspace gas composition was shown in Table 1. The substrate and air bags were replaced every 36 h, and the obtained liquid and gas sample were measured.
Except for 0% H2 level, the variation on hydrogen volume of other levels was lower than that in Group 1. There were some biochemical reactions consume hydrogen, including homoacetogenesis, HVa reaction, Propanol reaction, etc. Reductive deamination from sole amino acid is favorable and is hydrogen consuming (Dong et al., 2009). Since the only difference between the setting factors of Groups 1 and 2 was carbon dioxide and nitrogen in headspace of fermenter, it is inferred that the main cause of this phenomenon is the homoacetogenesis, which produced HAc with carbon dioxide and hydrogen as raw materials. It could be seen from the gas composition of Group 3 that the sparging of N2 was favorable for the formation of carbon dioxide; as the sparging rate increased, the volume of carbon dioxide changes were 94 ml, 144 ml, 214 ml and 295 ml, respectively, and the carbon dioxide content was 2.98% rises to 8.83%, indicating that the gas sparging rate had a great influence on the formation of carbon dioxide. The variations of hydrogen volume were 70 ml, 93 ml, 68 ml and 46 ml, respectively. This means that hydrogen production can be promoted at a nitrogen rate of 20 L/d. The blowing of nitrogen can cause a certain agitation and promote the escape of gas. But too fast flow is not conducive to the stability of the strain.
2.3. Analytical methods
3.2. VFAs concentration
Solluted Chemical oxygen demand (SCOD), VS and TS were determined according to standard methods (APHA, 1998). The soluble protein was quantified by the Bradford method using bovine serum albumin as the standard (Noaman and Coorssen, 2018). Volumetric biogas composition, including CH4, CO2, N2, and H2 was analyzed using a gas chromatograph (FULI; GC9790II; China) equipped with a thermal conductivity detector and packed column (Porapak Q column [3 m × 3 mm] cascading with a TDX-01 column [2 m × 3 mm]). The carrier gas was helium. The injector, oven, and detector temperatures were 150, 200 and 120 °C, respectively. Volumetric hydrogen and methane yields were calculated by multiplying the total yield by the corresponding volume percentages. The VFAs, including acetic acid (HAc), propionic acid (HPr), isobutyric acid (IBu), butyric acid (HBu), Isovaleric acid (IVa) and valeric acid (HVa) were measured using the same gas chromatograph equipped with a flame ionized detector (FID) and column detector (Kromat, KB-Wax; 30 m × 0.32 mm × 0.33 mm). The carrier gas was nitrogen at a flow rate of 75 ml/min. The inlet and detector temperatures were both 250 °C. The oven temperature was originally 80 °C and was increased to 200 °C at a rate of 20 °C/min.
The VFAs concentration in the fermentation vessel was obtained by recording the average value of the stable period, as shown in Fig. 3. In Group 1, the graph revealed that the VFAs concentration fluctuated all through. In 0% H2 level, the VFAs concentration was the lowest (5873.972 mg/L) among the five levels. Then it rose in 25% H2 level and dropped in 50% H2 level. The peak concentration (6554.295 mg/L) of VFAs was occurred at 100% H2 level. The trends for the concentration of HAc, IVa, IBu and HBu were similar, and this trend was similar to VFAs concentration. HPr and HVa did not change much in different conditions. According to Daltons law of partial pressures, the final PH2 were 0.018 atm, 0.203 atm, 0.441 atm, 0.645 atm and 0.793 atm, respectively. Many opinions believed that low PH2 favored the formation of acids (Zhou et al., 2017; Sarkar et al., 2017). But the observed phenomena were somewhat different from this view. Therefore, we had to consider the effects of homoacetogenesis and carbon dioxide partial pressure (PCO2 ). The PCO2 mainly acted on the homoacetogenesis, but we cannot estimate its other effects. Kim et al. (2006) reported that high PCO2 had little effect on H2-producing bacteria but inhibitory effect on other microorganisms such as acetogens and lactic acid bacteria which were competitive with H2-producing bacteria. The mechanism for this effect was not clearly stated. Eq. (2) is the reaction involved in acetate production without H2 generation, which are supported by the existence of acetate-producing bacteria which could consume H2 and CO2.
Table 1 Headspace composition.
1 2 3 4 5
Group 1
Group 2
Group 3 (N2)
0%H2 + 100%CO2 25%H2 + 75%CO2 50%H2 + 50%CO2 75%H2 + 25%CO2 100%H2 + 0%CO2
0%H2 + 100%N2 25%H2 + 75%N2 50%H2 + 50%N2 75%H2 + 25%N2 100%H2 + 0%N2
0 L/d 20 L/d 40 L/d 60 L/d –
3. Results and discussion 3.1. Analysis of headspace gas
4H2 + 2CO2 → CH3 COOH + 2H2 O( T= 37°C, ΔG′ = −74.54 kJ)
Table 2 showed the volume and composition of the headspace, all data taken from the mean value of the stable period. The volume of the headspace in Group 1 and 2 decreased slightly, but did not exceed 8%, while a small amount of gases was produced in Group 3. In Group 1, the percentage of hydrogen changed little except 100% H2 level. From 0% to 100% H2 level, the variations on hydrogen volume were 60 ml, −114 ml, −105 ml, −198 ml and −389 ml, respectively. The hydrogen gas volume decreases except for 0% H2 level, and with the increase of hydrogen percentage, the reduction of hydrogen also increased. The change of carbon dioxide is contrary to that of hydrogen, the variations on carbon dioxide volume were −288 ml, −162 ml, −122 ml, −11 ml and 284 ml, respectively. A similar situation occurred in Group 2. The small change in the percentage of gas content is in line with expectations, as this avoided the impact of excessive changes in gas composition on the test process. But in Group 2, the volume of remaining gas is slightly higher than Group 1. From 0% to 100% H2 level, the variations of hydrogen volume were 86 ml, −68 ml, −94 ml, −129 ml and −348 ml, respectively.
(2)
Δr Gm (T ) = △r Gmθ (T ) + RTlnQ Q=
[c (CH3 COOH )/ c θ] [PH2/ pθ ]4 [PCO2/ pθ ]2
Under standard conditions, the Gibbs free energy of the reaction is negative, which means that the reaction is easier to occur in the fermentation. △r Gmθ (T ) and Q reflected the influence of temperature and pressure on the reaction, respectively. As the fermentation proceeded, when the PH2 and PCO2 were reduced to a low enough level or the HAc concentration was high enough, the reaction will reach equilibrium, and the effect of PH2 is greater than PCO2 . As H2 level increased from 0% to 50%, the balance between PH2 and homoacetogenesis occurred in this range. To better determine when this balance occurred, it should be analyzed in conjunction with the results of Group 2. Between 0% and 50% H2 levels in Group 2, the concentration of HAc fell below 25% H2 level and began to make a recovery. In Fig. 4, through linear fitting, it 3
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Table 2 The biogas volume and composition. Component
Volume (ml)
Group 1 (%)
Volume (ml)
Group 2 (%)
Volume (ml)
Group 3 (%)
1
H2 N2 CH4 CO2
2823
2.11 1.83 0 96.06
2974
2.90 94.57 0 1.53
3165
2.22 94.80 0 2.98
2
H2 N2 CH4 CO2
2759
23.05 1.28 0 75.67
2824
24.16 71.38 0 4.46
3237
2.88 92.68 0 4.44
3
H2 N2 CH4 CO2
2788
50.05 0.52 0 49.43
2892
48.60 90.28 0 1.62
3282
2.07 91.41 0 6.52
4
H2 N2 CH4 CO2
2804
73.19 0.46 0 26.35
2872
73.84 22.86 0 3.30
3341
1.37 89.80 0 8.83
5
H2 N2 CH4 CO2
2901
90.01 0.18 0 9.81
2944
90.07 2.28 0 7.65
The deviation of test results is no more than 5%.
was found that the equilibrium point is between 25% and 50% H2 levels, and the PH2 was about 0.268 atm. Therefore, we could conclude that the promotion of low PH2 had been offset when the PH2 was greater than 0.268 atm. The fermentation medium can be easily supersaturated with H2 due to liquid-to-gas mass transfer limitation (Ljunggren et al., 2011; Zhang et al., 2012), this may be one of the reasons why PH2 promotes acid production at extremely low pressure. In Group 2, the trend of VFAs concentration declined below 25% H2 level and then rises. The maximum VFAs concentration was found in 100% H2 level (6331.405 mg/L); and the minimum concentration was found in 25% H2 level (5849.187 mg/L), which was different from Group 1. The concentration changes of HAc and HBu were basically the same as that of VFAs. There was no significant difference in IBu, HPr, IVa and HVa concentration. Compared with Group 1, the main difference was HAc. In Fig. 4, we found that in the same H2 level, the HAc concentration of Group 1 was 2–14% higher than that in Group 2. With the increase in PH2 and the decrease of PCO2 , the concentration of HAc became higher. And in 25% H2 level, CO2 had the greatest enhancement effect. As the PH2 continued to rise, the HAc concentration increased, but the difference in HAc concentration of the two groups becomes smaller, which indicated that the influence of PH2 on homoacetogenesis was greater. The PH2 in each level was 0.026 atm, 0.213 atm, 0.428 atm, 0.650 atm and 0.793 atm, respectively, which was similar to Group 1. So the main reason for the difference of acid concentration between Group 1 and 2 came from CO2. In Group 1, the PCO2 were 0.846 atm,
Fig. 4. Concentration of acetate in Group 1 and 2 with its fitting curve.
0.666 atm, 0.435 atm, 0.232 atm and 0.086 atm, respectively. But in Group 2, the PCO2 was very low, ranging from 0.013 to 0.067 atm. The PCO2 will directly affect the effect of homoacetogenesis and change the metabolic distribution. High PCO2 could significantly increase HAc concentration.
Fig. 3. VFA concentration in the reactor (Group 1: H2 + CO2, Group 2: H2 + N2, Group 3: N2, the deviation of test results is no more than 5%). 4
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In Group 3, it is observable that the VFAs concentration of Group 3 is significantly lower than that of Group 1 and 2. The main sources of this gap were acetate and butyrate. Besides, the concentration of IVa was higher, which may be the reason for the low concentration of acetate and butyrate. With the increase in flow rate, the concentration of VFAs fluctuated all through, and kept the maximum when the flow rate was 0 L/d, reaching 5501.806 mg/L. The main difference between the four levels was HBu. For example, at 0 L/d, the concentration of HBu was 1688.053 mg/L, and at 60 L/d, it was 1192.852 mg/L, which decreased by 29.38%. As the sparging rate increased, the trends of HPr, IBu, HBu, IVa and HVa concentration were the same, rising during 20–40 L/d but falling during 0–20 L/d and 40–60 L/d. HAc concentration was first decreased and then rises; no more than that, there is little difference between 0 L/d and 60 L/d (about 1870 mg/L), and between 20 L/d and 40 L/d (about 1700 mg/L). Different from other level, there is no artificial mixing process in the headspace air of 0 L/d, which may be one of the reasons for the change of acid production pathway. Gas sparging can cause sludge floating, gas escaping, etc. In brief, changes in the headspace can affect the concentration and distribution of VFAs, and even the metabolic pathways of microorganisms.
69–77. Alibardi, L., Muntoni, A., Polettini, A., 2014. Hydrogen and waste: illusions, challenges and perspectives. Waste Manage. 34, 2425–2426. APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association/American Water Works Association/Water Environment Federation, Washington, DC, USA. Bai, M., Cheng, S., Chao, Y., 2004. Effects of substrate components on hydrogen fermentation of multiple substrates. Water Sci. Technol. 50, 209–216. Dong, L., Zhenhong, Y., Yongming, S., Xiaoying, K., Yu, Z., 2009. Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation. Int. J. Hydrogen Energy 34, 812–820. Herman, R., Gao, Y., Storer, N.P., 2006. Acid-induced unfolding kinetics in simulated gastric digestion of proteins. Regulatory Toxicol. Pharmacol. Rtp 46 (1), 93–99. Hua Yan, B., Selvam, A., Wong, J.W.C., 2017. Influence of acidogenic headspace pressure on methane production under schematic of diversion of acidogenic off-gas to methanogenic reactor. Bioresour. Technol. 245, 1000–1007. Kim, D.H., Han, S.K., Kim, S.H., Shin, H.S., 2006. Effect of gas sparging on continuous fermentative hydrogen production. Int. J. Hydrogen Energy 31 (15), 2158–2169. Lee, K.S., Tseng, T.S., Liu, Y.W., Hsiao, Y.D., 2012. Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy. Int. J. Hydrogen Energy 37 (20), 15556–15562. 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 (1), 83–99. Liu, Y., Wang, Y., 2016. Directional enhancement of fermentative coproduction of hydrogen and acetic acid from glucose via control of headspace pressure. Int. J. Hydrogen Energy 42 (7). Liu, H., Wang, J., Liu, X., Fu, B., Chen, J., Yu, H.Q., 2012. Acidogenic fermentation of proteinaceous sewage sludge: effect of ph. Water Res. 46 (3). Ljunggren, M., Willquist, K., Zacchi, G., Van Niel, Ed W.J., 2011. A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus. Biotechnol. Biofuels 4 (1), 31. Noaman, N., Coorssen, J.R., 2018. Coomassie does it (better): a Robin Hood approach to total protein quantification. Anal. Biochem. 556, 53–56. Pham, T.P.T., Kaushik, R., Parshetti, G.K., 2015. Food waste-to-energy conversion technologies current status and future directions. Waste Manage. 38, 399–408. Piotr, S., Krzysztof, P., Jan, C., Jolanta, B., 2017. Alternative utilization of protein-rich waste by its conversion into biogas in co-fermentation conditions. Pol. J. Environ. Stud. 26 (3), 1225–1231. Ramsay, I.R., Pullammanappallil, P.C., 2001. Protein degradation during anaerobic wastewater treatment: derivation of stoichiometry. Biodegradation 12 (4), 247–256. Sarkar, O., Butti, S.K., Venkata Mohan, S., 2017. Acidogenesis driven by hydrogen partial pressure towards bioethanol production through fatty acids reduction. Energy 118, 425–434. Shen, D.S., Yin, J., Yu, X.Q., Wang, M.Z., Long, Y.Y., Shentu, J.L., Chen, T., 2017. Acidogenic fermentation characteristics of different types of protein-rich substrate food waste to produce volatile fatty acids. Bioresour. Technol. 227, 125–132. Sostaric, M., Klinar, D., Bricelj, M., Golob, J., Berovic, M., Likozar, B., 2012. Growth, lipid extraction and thermal degradation of the microalga Chlorella vulgaris. New Biotechnol. 29, 325–331. Valero, D., Montes, Jesús A., Rico, José Luis, Rico, C., 2016. Influence of headspace pressure on methane production in biochemical methane potential (BMP) tests. Waste Manage. 48 (1), 193–198. Xia, A., Cheng, J., Lin, R., Liu, J., Zhou, J., Cen, K., 2013. Sequential generation of hydrogen and methane from glutamic acid through combined photo-fermentation and methanogenesis. Bioresour. Technol. 131, 146–151. Yan, B.H., Selvam, A., Xu, S.Y., Wong, J.W.C., 2014. A novel way to utilize hydrogen and carbon dioxide in acidogenic reactor through homoacetogenesis. Bioresour. Technol. 159, 249–257. Yang, X., Choi, H.S., Park, C., Kim, S.W., 2015. Current states and prospects of organic waste utilization for biorefineries. Renew. Sustain. Energy Rev. 49, 335–349. Yang, Z., Guo, R., Xu, X., Fan, X., Luo, S., 2011. Hydrogen and methane production from lipid-extracted microalgal biomass residues. Int. J. Hydrogen Energy 36, 3465–3470. Yin, B., Liu, H.B., Wang, Y.Y., Bai, J., Liu, H., Fu, B., 2016. Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: protease catalysis of refractory protein. Bioresour. Technol. 203, 124–131. Zhang, F., Zhang, Y., Chen, M., Zeng, R.J., 2012. Hydrogen supersaturation in thermophilic mixed culture fermentation. Int. J. Hydrogen Energy 37 (23), 17809–17816. Zhou, M., Zhou, J., Tan, M., Du, J., Yan, B., Wong, J.W.C., Zhang, Y., 2017. Enhanced carboxylic acids production by decreasing hydrogen partial pressure during acidogenic fermentation of glucose. Bioresour. Technol. 245, 44–51.
4. Conclusions The different headspace air has a significant impact on the fermentation process. PH2 , PCO2 and homoacetogenesis have a greater impact on the distribution of acid production. When the PH2 and PCO2 are high, the homoacetogenesis will be promoted, which causing an increase in acid concentration. But the effect of PH2 is greater than that of PCO2 . Homoacetogenesis play a role part when the PH2 was greater than 0.268 atm. Addition of CO2 is beneficial to HAc production, the PCO2 will directly affect the effect of homoacetogenesis and change the metabolic distribution. When the PCO2 is 0.666 atm, the promotion effect reaches the maximum. High PCO2 could significantly increase HAc concentration. In Group 3, the concentrations of acids are both lower than Group 1 and 2. Sparging way is not conducive to acid formation, but conducive to gas production. Accordingly, if we aim to produce acid, the fermentation system can be divided into two phases of hydrolysis acid production and acetic acid production, and the two-phase acid production is promoted when the gas for hydrolyzing acid is introduced into the second phase. Acknowledgements This work was financially supported by grants from National Natural Science Foundation of China (no. 51606083), Fundamental Research Funds for the Central Universities of China (no. 2662008JC006), and Youth scholar promotion project of Dabei agricultural fund (2017DBN007). References Alibardi, L., Cossu, R., 2015. Effects of carbohydrate, protein and lipid content of organic waste on hydrogen production and fermentation products. Waste Manage. 47 (Pt A),
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