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Reflux of acidizing fluid for enhancing biomethane production from cattle manure in plug flow reactor
Bioresource Technology 284 (2019) 248–255
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Reflux of acidizing fluid for enhancing biomethane production from cattle manure in plug flow reactor
T
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Lili Dong, Guangli Cao , Jiwen Wu, Shanshan Yang, Nanqi Ren State Key Laboratory of Urban Water Resources and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Reflux acidizing fluid Cattle manure Biogas Plug flow reactor Microbial community succession
The performance and microbial community succession of a 36 L working volume plug flow reactor was evaluated for treated cattle manure at an organic loading rate of 2.67 g-TS/L/day, a temperature of 38 °C ± 1 °C, and a hydraulic retention time of 18 days. A reflux of acidizing fluid effectively enhanced anaerobic digestion performance and promoted optimization of microbial community structure. The average biogas volume production rate was 1.08 L biogas/L reactor, which was 116.5% higher than the control without reflux of acidizing fluid. The specific qmethane production yield and methane content reached 0.204 L/g VS and 70%. Moreover, methane yield achieved 0.34 m3/kg removal COD with a COD removal of about 70.56%. The bacteria genera Christensenellaceae, Bacteroidales, vadinBC27, Ruminococcaceae and Treponema_2 were further enriched. Methanosarcina became the dominant methanogen in the whole PFR operation process. This study offers new opportunities for producing renewable energy from enhanced cattle manure biodegradability.
1. Introduction Animal waste is one of the most abundant waste fractions generated worldwide. The amount of manure produced increases daily as the number of livestock and poultry breeding industry grows. Traditionally, manure management has relied on the basic agronomic approach of storage and field spreading for production of forage crops. Although effective for nutrient recycling, these systems can be odor intensive and have adverse environmental impacts in terms of terrestrial eutrophication and greenhouse gas emissions (CHG) (Muhammad et al., 2016; Sommer et al., 2009). Anaerobic digestion (AD) is considered an economical and effective method for the treatment of animal wastes (Cheng et al., 2011; Lansing et al., 2008). In spite of low biogas yields due to its high fiber content, cattle manure is an excellent ‘carrier’ substrate for biogas production in AD of concentrated waste – which would be difficult to treat separately – because of its high water content, high buffering capacity and wide variety of nutrients, which are necessary for optimal bacterial growth (Kaparaju et al., 2009; Angelidaki and Ellegaard, 2003). Various authors have studied techniques to improve manure’s biogas production, including development of low-cost digesters, key operating parameters, and pretreatment technique e.g. (Khalid et al., 2011; Ballyk and Smith, 1999). The sludge refluxing method is an effective control measure mainly used to reduce environmental impact and operate costs and to simplify
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management in urban sewage treatment. Recently, biogas slurry refluxing has been used as a technical method to improve yields in biogas projects (Shahriari et al., 2012; Hao et al., 2008). However, some problems may occur after the long-term operation in industrial application, such as ammonia accumulated, volatile fatty acids (VFAs), nonbiodegradable intermediates, and other inhibitory substances (Hu et al., 2014; Wang et al., 2018). The accumulation could result in changes to the anaerobic environment and inhibition of microbial activity, with the potential to cause complete malfunction of the anaerobic digestion system (Mshandete et al., 2004). Microorganisms are the core of AD, and as this biochemical process involves a great variety of microbial groups, the success of this anaerobic digestion process depends crucially on their growth and metabolism (Bocher et al., 2015; Wang et al., 2018). The plug-flow reactor (PFR) is often referred to as a low-cost anaerobic digester. The contents are not mixed, and continuous management or internal heating is not required when located in a tropical climate (Lansing et al., 2008). The PFR’s separation into acidogenic and methanogenic phases along the flow path of the reactor improves reactor stability and treatment efficiency (Yue et al., 2011; Daniel et al., 2013). As known the limiting step of anaerobic fermentation is hydrolysis, especially for substrates with high lignocellulosic content. So the PFR maybe meet the requirements of staged regulation reactor to improve hydrolysis efficiency. In addition, initial investments for PFR
Corresponding author. E-mail address: [email protected] (G. Cao).
https://doi.org/10.1016/j.biortech.2019.03.092 Received 1 February 2019; Received in revised form 15 March 2019; Accepted 17 March 2019 Available online 19 March 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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investigation.
installation and start-up are typically lower than those for other kinds of reactor configurations because they do not have internal moving parts and usually require less computer control and human intervention. Moreover, a PFR avoids the ‘short-circuit’ phenomenon because it involves the first-in and first-out material and enough hydraulic retention time (Rowse, 2011; Patinvoh et al., 2017; Kothari et al., 2014). However, anaerobic treatment of animal wastes in laboratories, at full scale and under different operating conditions, commonly employs completely stirred tank reactors (CSTR) (Khalid et al., 2011). Although low-cost PFRs have been developed, most of these have focused on design, construction, and optimization. Rowse (2011) has demonstrated a methane yield of cattle manure and straw in a horizontal PFR of 0.163 L CH4/g VS added at a hydraulic retention time (HRT) of 40 d and a temperature of 37 °C. Subsequently, Daniel and Massé et al. (2013) investigated a PFR treatment of pig manure at a low temperature (25 °C) and HRT of 67 d, obtaining specific methane yields ranging from 0.1486 L CH4/g TCOD. However, to the authors’ best knowledge, there is a lack of research on the effect of refluxing acidizing fluid (RAF) on the AD process, especially involving cattle manure as the PFR substrate. Moreover, there have been few studies on the relationship between the transformation process of the substrate and the succession process of microbial communities. The main objective of this paper was to show the profitability and advantages in the design of a PFR with RAF to degrade cattle manure at a lab-scale for 90 days operation. The purpose was to reflect the operation performance of the digester and provide more accurate technical data for guided industrial application. During the digester operation process, 16S rRNA gene sequencing was used to characterize the dynamics of the microbial community structure evolution from operation phase to RAF operation phase. Meanwhile the nutrient of the chemical oxygen demand (COD) removed efficiency, total solid (TS) and volatile solid (VS), pH, biogas production and the content of biogas were determined. This gave a unique opportunity to deep insight the influences of RAF on substrate conversion and microbial community in PFR apparently operating close to the edge of stability.
2.2. Reactor configuration and operation 2.2.1. Reactor configuration A horizontal PFR (Fig. 1) for continuous anaerobic digestion cattle manure process was constructed with transparent Plexiglas sheets with a thickness of ten millimeters in a rectangular shape, which was designed and made at the Harbin Institute of Technology, Harbin. The PFR had a total length of 3 m, which was composed of two 1.5 m reactors in parallel series, and a width of 0.1 m and a height of 0.15 m, 0.03 m for gas space in height (which including the total height), with a capacity of 45 L and an active working volume of 36 L. The PFR was divided into 10 equal compartments, they were K1, K2, K3, K4, K5, K6, K7, K8, K9 and K10 from the inlet to the outlet. The inlet and outlet were on the same side. The reflux port was located in the middle of the K2. The top Plexiglas supported temperature measuring ports, sampling ports and gas collection ports. The external PFR was set up circulating hot water in the space between the reactor Plexiglas walls to ensure the constant temperature required for the digester. 2.2.2. Reactor start-up and operation The PFR was operated with 5% by wt of TS (equivalent to about 50,000 mg/L COD). In order to allow acclimation period for the anaerobic microorganisms within digester, gradual and careful changes in the environment was employed. Stable digester temperature was maintained at 38 ± 1 °C by circulating hot water installation. Prepared substrate was semi-continuously fed into the digester one time a day by pumping the substrate from the homogenizer. Operating pressure was around 1 atm. Pressure from the new feed and biogas inside the reactor pushed out the effluent from liquid surface through the gas collection ports on the top of digester. The outflows were collected in effluent bucket. Biogas from the digester flowed further to gas bags for measuring biogas production and compositions. Biogas production was collected by gas collecting bag from every gas collection hole and measured by dewatering method reversible cycle and registration. The whole fermentation operation process can be summarized into three different phases: start-up (from 1 to 20 day), operation phase (21–40 day) and RAF operation phase (41–90 day). During the RAF operation phase, half of the daily feed amount from the reflux port was added into the substrate before feeding. Experiments were carried out using progressive organic loading rate during start-up stage. The loading rate of 0.67 g-TS/L/day (OLR1) continued only about 2 days. Then it gradually increased to around 1.33 g-TS/L/day (OLR2) in the next 3 days and further increased to 2 g-TS/L/day (OLR3) in the next 6 days. The 2.4 g-TS/L/day (OLR4) continued 5 days. Then the organic load rate was added to the design value 2.67 g-TS/L/day (OLR5) after 16 days and remained at the same level until the end of start-up period and the further operation. From day 21 to operation end, the HRT was 18 days. The inoculum was added at 80% (v/v). Before feeding, an equal amount of digestion was withdrawn.
2. Materials and methods 2.1. Substrate and inoculum characteristics Substrate used in the experiment was cattle manure from WONDERSUN Green Hill Foot Milk Source Base, Harbin. The fresh cow manure was collected in a closed container, stored in the 4 °C for further use and replaced every seven days. The manure was mixed thoroughly and heated 36–40 °C before use. Liquid inoculum used in this research was obtained from a large-scale PFR project of the Modern farming group of the Shuangcheng 3 MW cow manure disposal, which had been running for more than three year under mesophilic conditions (Dong et al., 2019). Table 1 illustrates the most important physical and chemical characteristics of cattle manure and inoculum used in this Table 1 Characteristics of the cattle manure and inoculum. Parameter
Substrate
Inoculum
pH ORP (mv) TS (%) VS (%) TN (mg/L) TP (mg/L) COD (mg/L) VFA (mg/L)
7.76 ± 0.01 −325.00 ± 9.1 15.35 ± 2.35 13.51 ± 0.53 1184.70 ± 25.82 575.21 ± 13.35 159050.00 ± 15.43 1534.60 ± 5.03
7.67 ± 0.01 −221 ± 5.35 3.82 ± 0.01 1.80 ± 0.01 11.37 ± 4.23 85.62 ± 0.46 27975 ± 9.86 234.21 ± 1.45
2.2.3. Analytical methods In the process of starting and stabilizing, inflow and outflow samples were collected from inlet and outlet of the reactor every day. Samples from the K2 and K6 sample holes of the reactor every 5–7 days. Then the pH, oxidation reduction potential (ORP), TS, VS, COD and contents of biogas (CH4 and CO2) were determined every day. The TS and VS were assayed according to Standard Methods (APHA, 1998). The pH and chemical oxygen demand (COD) were detected by HACHHQ40d multi and HACH-DRB 200, respectively. The total nitrogen (TN) and the total phosphorus (TP) were measured by analytikjena multi N/ C 2100s and Perkin Elmer Optima 8300, respectively. The oxidation reduction potential (ORP) was tested by C220-ORP (Shanghai Precision Instruments Co., Ltd). The gas products (CH4 and CO2) were determined by gas chromatography (GC, 7890A, Agilent Cooperation, USA).
VS, volatile solids; TS, total solids; COD, chemical oxygen demand; TN, total nitrogen; TP, total phosphorus; VFA, volatile fatty acids; ORP, oxidation reduction potential. 249
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Fig. 1. Schematic process flow diagram of PFR anaerobic fermentation system.
biogas production, which was only 4 L/d (0.12 L gas/L reactor) with the CH4 content was less than 45% (Fig. 2b). The result may a lag or adaptation period in which the methanogenic bacteria acclimatized to the new environment. Additionally, the accumulated air in the reactor was released by biogas production and emission to lead low CH4 content. In the following days, the biogas production was observed to gradually increase from 5.76 L/d (0.17 L biogas/L reactor) to 21.40 L/d (0.63 L biogas/L reactor) during 4 to 20 d, and the CH4 content maintained a relatively stable value of 56.20% ± 5.13%. This result compared favorably with Jagadish et al. (1998) who reported the biogas production of 0.5 m3 gas/m3 reactor when the digestion of leaf was investigated in PFR, suggesting that the biogas slurry started adapting to the new environment and the digester successfully started up. After successful start-up of the digester, the digester entered operation phase. As shown in the Fig. 2a and b, the daily biogas production fluctuated due to fluctuation of daily OLR, from 14.69 L/d to 22.08 L/d during day 21 to day 40. The average value was 17.16 L per day (0.53 L biogas/L reactor) and the CH4 content maintained a relatively stable value of 57.01% ± 4.94%. This result was similar to or even higher than the average results reported in manure biogas production researches (Wang et al., 2017; Jagadish et al., 1998). The specific biogas yield and methane yield also have a small range of fluctuations, and the average specific biogas and methane yields were 0.15 ± 0.02 L/g VS and 0.10 ± 0.02 L/g VS, respectively (Fig. 2c), which are comparable to those reported by Abubakar and Ismail (2012) and Risberg et al. (2013). Moreover, the biogas production-based removal COD was around 0.39 m3/kg removal COD, with 0.22 m3/kg removal COD CH4 production.
2.2.4. DNA extraction and Illumina sequencing For insighting the change of microbial community succession before/after reflux acidifying liquid, samples (3–10 mL) from the K2 and K6 sample holes were taken over the period of operation phase and after adding reflux acidifying liquid operation phase with a sterilized sample spoon and stored in a 50 mL sterile plastic test tubes at −80 °C before went for DNA analysis. Microbial DNA was extracted from collected samples using the bacterial DNA mini kit (Watson Biotechnologies Co. Ltd., Shanghai, China) according to manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, USA), and DNA quality was checked by 1% agarose gel electrophoresis. Bacterial V3-V4 region of 16S rRNA gene was amplified using the forward and reversed primer 338F (5′- ACTC CTACGGGAGGCAGCAG-3′) and 806R (5′- GGACTACHVGGGTWTCTAAT-3′). Archaea V4-V5 region of 16S rRNA gene was amplified using 524F (5′-TGYCAGCCGCCGCGGTAA-3′). PCR products were purified using GeneAmp (9700 ABI, USA) PCR purification kit (Axygen Biosciences, Union City, CA, USA) and then went for Illumina sequencing platform. The resulted PCR products were quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s protocol. The sequences obtained from Illumina sequencing were analyzed following the pipelines of Quantitative Insights into Microbial Ecology (QIIME) software (www.microbio.me/qiime) as described by the Huang et al. (2015). Taxonomic classification of each phylotype was determined using the SILVA rRNA database project with over 97% of sequence similarity.
3. Results and discussion 3.2. Performance of PFR with RAF 3.1. Start-up and operation of the PFR 3.2.1. Daily biogas production Biogas production is an intuitive indicator for a digester that can reflect the performance of anaerobic digester efficiency and property. To investigate the effect of RAF on the digester operation process, the acidizing fluid from the reflux port was recirculated in the inflow substrate every day from day 41 to the end of operation. The daily biogas production and volume biogas production rate were illustrated
For an anaerobic reactor, a quick and stable start-up is very important. In this study, the start-up was carried out using progressive organic loading rates. Moreover, the inoculum was added at 80% (v/v) to improve the microbial content and shorten the start-up time. It was observed that the daily biogas production increased with the increasing of OLR (Fig. 2a). During the first 3 days of observation, there was less 250
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Fig. 2. Performance of PFR during the whole operation. (a) Daily biogas production and volume biogas production rate; (b) Variation of methane and carbon dioxide content in produced biogas from PFR; (c) Variation of specific biogas (methane) and biogas (methane) production based removal COD; (d) COD removal with different operation phase; (e) pH value of in/out flow and samples of PFR.
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initial days, so the high removal rate of COD was obtained (Fig. 2d). From day 17, the organic load rate was added to the design value 2.67 g-TS/L/day, and the COD removal efficiency retained around 45% during the operation phase. After added RAF, COD removal fluctuated from day 42 to day 72, but overall, COD removal improved. After day 72, COD removal stabilized at about 70.56%, showing that anaerobic fermentation had entered its stationary phase. The fluctuations of COD removal efficiency could be due in part to the substrate of PFR being first in and first out, meaning that the outlet situation of one day was affected by the inlet substrate of the previous 18 days. Also, the cattle manure used in this research was collected every week to ensure that its biodegradable portions weren’t digested due to long-term storage, which meant that input COD fluctuated. Overall, the average COD removal rate increased 25.6% compared with the operation phase, indicating that RAF contributes to the substrate degradation. This also implies that the fermentation system had buffering capacity for unstable inlet COD and thus has potential for industrial application, because unstable COD loading is widespread in actual biogas production on an industrial scale.
(Fig. 2a). It was observed that biogas production significantly increased from day 42. In the following 19 days, daily biogas production continued increase until day 61. After that, biogas production stayed around 37.15 L/d (1.08 L biogas/L reactor) until the digester stopped operation. This result showed a 116.5% increase in biogas production after RAF compared with operation phase. In our previous work, a large-scale PFR project of the Modern farming group of the Shuangcheng 3 MW cow manure disposal, which had been running for more than three year under mesophilic conditions, the volume biogas production rate was 0.58 L biogas/L reactor (Dong et al., 2019). And the operation parameters (refer to fermentation temperature, residence time, reactor type, etc.) of the project were basically the same as this study. Compared with industrial engineering, the result of this study increased 86.21%. This increase was satisfactory, and indicated that the RAF enhancing the raw material biogas production efficiency. 3.2.2. Methane and carbon dioxide content of produced biogas Usually, CH4 and CO2 are the two main components present of biogas. CH4 is the energy product, so CH4 content in biogas is a key index for industrial production of biogas. A higher CH4 content means more valuable biogas production. The content of CH4 in biogas significantly increased during the RAF operation phase (Fig. 2b). The CH4 content was between 45.98% and 69.51% with an average at around 65.2%, more than 8.19% higher than that in the operation phase. Moreover, the maximum CH4 content was close to 70%. This result was similar to that of Yang et al. (2015), who reported that the CH4 content of biogas production from anaerobic digestion was about 53–70%.
3.2.5. pH During AD process, pH is an important parameter affecting the growth of microbes during anaerobic digestion (Yadvika et al., 2004). Inflow and outflow pH were measured every day to evaluate PFR operation process, which partly reflects the conditions of the fermentation system (Fig. 2e). After start-up, the pH of inflow material varied widely from 6.99 to 7.51 between operation phase and RAF operation phase. The main reason was that the pH of the fresh cattle manure collected every week fluctuated greatly. While, there were a small fluctuation for the pH of outflow overall the whole operation process and the pH stable at 7.54 ± 0.12. It indicated that the anaerobic fermentation is always in a normal state after start-up. For the pH change in the digester, K2 and K6 samples of the digester representing the state of the acidogenic and methanogenic phase respectively. It can be seen from Fig. 2e, the pH for the K2 sample reduced significantly when the RAF was added, and it tended to be stable and maintained at around 6.28 after 30 days. This indicates that hydrolytic acidification was increased after RAF. However, the pH of the K6 sample showed small fluctuations (from 7.31 to 7.6) between operation phase and RAF operation phase. According to the report, most anaerobic bacteria, including methane-forming bacteria, perform well within a pH range of 6.8–7.2 (Chandra et al., 2012). In this study, biomethane production remained at a good level at a pH between 6.27 and 7.60. This suggests that the PFR has a wider resistance to the pH of the substrate which would have been very beneficial for anaerobic fermentation to increase biogas production. To summarize, based on system stability, methane production, substrate utilization and pH, RAF could be considered a suitable control measure for enhancing the AD of cattle manure in PFR. In addition, PFR is probably a highly efficient and economic configuration for applying in waste treatment with high lignocellulose content.
3.2.3. Specific biogas and methane production yield Besides biogas production, specific biogas yield (or methane yield) is also an important index in industrial applications (Marimuthu and Kirubakaran, 2015). This index captures the biogas (methane) production per unit VS and per unit removal COD in the case of cattle manure substrate throughout this long 90-day fermentation process (Fig. 2c). During the RAF operation phase, the specific biogas (methane) production yield increased with the fluctuation from day 42. With the RAF continuous operation, the specific biogas (methane) production yield reaching a maximum of 0.354 L/g VS and 0.20 L/g VS on the 59th day which increased nearly 50% than non-RAF. In the following days, the biogas and methane production yield maintained at 0.329 ± 0.016 L/g VS and 0.204 ± 0.015 L/g VS, respectively. Similarly, the yield of biogas production-based removal COD increased significantly as biogas production increased. Although there were large fluctuations, a very satisfactory result was obtained after RAF, the average biogas production-based removal COD was 0.58 m3/kg removal COD and 0.34 m3/kg removal COD CH4 production, which were 48.7% and 54.5% higher than that in the operation phase without RAF. Laboratory-scale studies with upflow anaerobic sludge blanket (UASB) reactor have demonstrated that anaerobic treatment of manure at mesophilic with the production of biogas was 0.20 m3/kg COD removed (Castrillón et al., 2002). Additionally, CSTR, in comparison, produced a biomethane yield of about 0.224 m3/kg TS (Ismail et al., 2012). Meanwhile, the 0.34 m3 CH4 /kg removal COD obtained in this study nearly reached the 0.35 m3 theoretical value. This value is very attractive for industrialization. Thereby, it is very clear that the RAF measure is potentially effective for enhancing biogas and methane production yields from cattle manure waste.
3.3. Structure of microbial community succession A flexible community structure maintains a stable ecosystem function. To better understand the correlation between performance and microbial community structure succession established during the whole fermentation process, bacterial and archaeal communities were analyzed through Illumina sequencing. For stable performance, the microbial community in the reactor should be dynamically balanced. Hence, the samples were taken from K2 and K6 of the 21nd of operation phase and 68th of RAF operation phase. As a result, the samples deviated slightly, but indeed reflected the overall trend.
3.2.4. COD removal COD removal is one important index of reflect the efficiency and stability of fermentation systems. Usually, for an anaerobic fermentation system of cattle manure, COD removal is 51–79% (Rowse, 2011). During the start-up, for the first three days of fermentation, the performance of COD removal was very high, about 90%, but this value gradually decreased to 72.9% in the next days (from 4th to 17th), suggesting that the digestion process properly at the low OLR in the
3.3.1. Comparative analysis of bacterial communities across samples Compared with operation phase, different bacterial community structures were discovered in the RAF operation phase, and their ratio 252
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Fig. 3. Phylum level distribution of bacterial (a) and archaeal (b) community in digeater’s samples at operation phases and RAF operation phase.
This indicated that in the PFR anaerobic digester, most biodegradable substances have completed the process of hydrolytic acidification in the front of the digester, and the back of digester mainly hydrolyzed and acidified substances that are difficult to biodegrade. Xie et al. (2014) reported the genus vadinBC27_wastewater-sludge_group degrades difficult-to-degrade organic substances. It has also been suggested that Ruminofilibacter and Ruminococcaceae play important roles in cellulose hydrolysis in the rumen (Ransom-Jones et al., 2012). According Evans et al. (2015), the Turicibacter group can positively in fluence the host animal by improving the digestion of complex organic matter such as cellulose. Furthermore, recent reports of the 16S rRNA gene and ribosomal intergenic spacer analysis data indicate the presence of Turicibacter bacteria in the rumen and faeces of cattle and pigs, and in rat and goat hindguts (Zhou et al., 2016). A further change in the bacterial community structure was recorded when the RAF was applied to the digester. The differences were as significant as those observed when the substrate was pushed to the outlet. Among Firmicutes, species as Christensenellaceae were predominantly present in the RAF operation phase, 11.17% in K2 and 11.94% in K6. Meanwhile, Bacteroidales, vadinBC27_wastewater-sludge_group, Ruminococcaceae and Treponema_2 were also prominent in K2. However, unlike K2 in the operation phase, the results of the RAF operation phase were different, the dominant bacteria was Ruminofilibacter (16.36%), accompanied mainly with Ruminococcaceae, vadinBC27_wastewater-sludge_group and Treponema_2. Some studies have been conducted on related rapid and highly efficient degradation of matter. We concluded that the Christensenellaceae genera can form a symbiotic association by highly efficiently converting the material of cattle manure, such as cellulose, hemicellulose and lignin, and that the Ruminofilibacter genus was closely related to the high-speed
among the groups varied considerably. From the Fig. 3a, it can be obtained that the Firmicutes and Bacteroidetes decreased in the RAF operation phase. While, the abundance of Spirochaetae increased by nearly 5 times. Additionally, Proteobacteria significantly increased by nearly 50%. Fibrobacteres, as one of the most important functional bacteria for lignocellulose degradation, increased by 13.4 times in the RAF operation phase. Besides, the other functional bacteria such as Synergistetes, Tenericutes and Chloribi also showed obvious increase in the RAF operation phase. The most visual changes to community structure are that several functional bacteria were obviously enriched in the PFR after RAF, while the other bacteria were reduced to a smaller proportion. Based on the biogas production results, it seems that the abundant functional microorganisms and the structures of specific microorganisms of these phyla were more functionally important for improved the digestion of cattle manure wastes in our experiments. Fig. 4a shows the genus classification of the bacterial community at different samples and phases from the PFR. The most dominant bacterial was Bacteroides in the operation phase. Meanwhile, there were other functional bacteria such as Peptostreptococcaceae, Paeniclostridium, Caproiciproducens, Hydrogenispora, Ruminiclostridium, Ruminococcaceae. The Bacteroides form a phylogenetically highly diverse group and are known as hydrolytic fermentative degraders of polymers in mainly anaerobic habitats (Li et al., 2014). Hernon et al. also reported similar findings when performing AD of carbohydrate-rich waste in a mesophilic anaerobic reactor. When the substrate was pushed to the outlet, some of bacteria that contribute to decomposing difficult-to-degrade organic substances appeared, such as Peptostreptococcaceae, Paeniclostridium, Ruminofilibacter, Ruminococcaceae, vadinBC27_wastewater-sludge_group, Turicibacter, norank_f__Draconibacteriaceae and Christensenellaceae, and the contents of these bacteria were no significant difference.
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Fig. 4. Genus level of bacterial (a) and archaeal (b) community in PFR’s samples at operation phases and RAF operation phase: the species, whose sums of percentage in all the samples are less than 1%, are classified as ‘the others’.
digester mainly used H2/CO2 to produce methane, a large amount of volatile acid accumulated. As the substrate was pushed to the outlet, a large amount of Methanosarcina and Methanosaeta was enriched with acetic acid as the methane bacteria metabolic substrate, so the methane bacteria metabolic substrate mainly used acetic acid in the back of the digester. Methanosarcina in AD reactors can be considered beneficial, since this quite robust archaeon is able to use different substrates. Thereby, multiple pathways for methane production because of its relatively high growth rate and ability to tolerate conditions inhibitory to other methanogens, such as the presence of ammonia or volatile acid accumulation (De Vrieze et al., 2012). However, Methanosarcina was interestingly significantly more abundant in the K2 sample of the RAF operation phase (an increase of 46.83%) accompanied with a 46.91% drop in Methanobrevibacter. It is likely that the proportion of Methanosarcina increased significantly after the acidizing fluid flow regulation maybe associate with a higher concentration of the acetic acid reflux group. At the same time, other hydrogenotrophic methanogens: Methanobacterium, Methanocorpusculum and Methanospirillum were enriched. The state of the community structure of K6 after RAF, there no significant change was observed. Therefore, the acetoclastic pathway was dominant in the RAF operation phase. Most interestingly, the total abundance of hydrotrophic methanogens was similar to methamphetamine. Ultimately, under the highly efficient biomethane-producing state of the digester, the microorganisms could achieved a state of balance to make the digester operate efficiently and steadily. We speculated that the RAF promoted the enrichment of Methanosarcina at the front of the digester, which could efficiently and timely consume the hydrolysate, thus improved the overall biogas production efficiency of the digester. To sum up, this research could provide deeper insights into microbial function in the PFR AD process. RAF increased the microorganism in the feed material, then increases the hydrolysis and acidification of the substrate and promotes the growth of methanogens. Ultimately, RAF can realize a balance of microbial hydrolysis and methane production and create high-efficiency biomethane production. The data obtained provide a basis for the further regulation and control of the complex interactions of the bacterial and archaeal communities in industrial full-scale mesophilic biogas plants.
degradation of cellulose. Ruminofilibacter hydrolyzed lignocellulose by secreting a large amount of cellulase and hemicellulase, and produced a lot of acetic acid and butyric acid, which were used by the methanogenic bacteria and the result was in agreement with Patinvoh et al. (2017). This result suggests that the addition of RAF not only increased the characteristic bacteria of hydrolysis acidification in the front of the digester, but also further increased the bacteria that highly efficiently digest difficult-to-degrade cellulose in the back of digester. Meanwhile, based on the results, the digester allowed the coexistence of more bacteria species during the RAF operation phase. In conclusion, the findings clearly demonstrate that in PFR fermentation of cattle manure, the reflux part of acidification liquid of the reactor can promote to increase the microorganism content of feed materials. At the same time, it can enhance the abundance of highly efficient cellulose-degrading bacteria. This is very beneficial to anaerobic fermentation and a main factor for obtaining higher biogas production yield.
3.3.2. Comparative analysis of archaeal communities across samples Understanding the taxonomic distribution and properties of methanogens allowed us to infer methanogenic pathway at different parts and phases of the digester. Euryarchaeota and Bathyarchaeota were the dominant archaea phyla in the operation phase and RAF operation phase (Fig. 3b). However, there were differences in these two operation phases. Euryarchaeota in the RAF operation phase was 15.44% higher than in the operation phase. On the contrary, Bathyarchaeota was much less present in the RAF operation phase. Additionally, a small amount of WAS2 presented after added RAF. From the results of archaea phyla, the RAF measure can promote the adjustment of the population structure of archaea in the system. Genus-level classification was conducted to further determine phylogenetic discrimination and possible functions of the archaeal members in the digester during different phases. The predominant genera were Methanobrevibacter (72.11%) and Methanocorpusculum (15.68%) in the K2 sample of the operation phase (Fig. 4b). Methanobrevibacter and Methanocorpusculum can use H2 /CO2 as a substrate of methanogenesis. Methanocorpusculum can also use parts of formic acid produced by methane. This suggests that methane mainly produced the H2 reduction of CO2 in the front of digester. In K6 of the operation phase, the dominant archaea were Methanosarcina with relative abundance of 35.12% and Methanobrevibacter with 23.20%. Also present were norank_p__Bathyarchaeota with 17.35%, Methanosaeta with 9.38% and Methanocorpusculum with 9.16%. Because the methanogens at the front of
4. Conclusions This study investigated RAF as one efficient control measure technology for enhancing biogas production and optimizing the microbial 254
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community structure in a PFR digester. Peak daily biogas production reached 39.32 L/day with 1.14 L biogas/L reactor and 0.329 ± 0.016 L/g VS with a CH4 content of close to 70% and a COD removal rate of 70.56% in the PFR during the RAF operation phase. Meanwhile, 0.34 m3/kg removal COD CH4 production was achieved. Many functional microorganisms enriched hydrolytic acidification, especially bacteria that digest difficult-to-degrade cellulose-for example, Christensenellaceae, Ruminofilibacter and Fibrobacteres. Methanosarcina was dominant during the RAF operation phase. Therefore, this technology has practical guiding significance for industrial applications.
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Acknowledgements This research was supported by National Natural Science Foundation of China (No. 31870110), Heilongjiang Province Foundation for Returness (No. JJ20170414), Special Fund for Scientific and Technological Innovation of Harbin (No. JJ20170385), Postdoctoral Scientific Research Developmental Fund of Heilongjiang Province (No. LBH-Q15054), and State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2018TS02). References Abubakar, B.S.U.I., Ismail, N., 2012. Anaerobic digestion of cow dung for biogas production. ARPN: JEAS. 7 (2), 169–172. Angelidaki, I., Ellegaard, L., 2003. Codigestion of manure and organic wastes in centralized biogas plants. Appl. Biochem. Biotechnol. 109 (1–3), 95–105. American Public Health Association (APHA), 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA, Washington, DC. Ballyk, M., Smith, H., 1999. Model of microbial growth in a plug flow reactor with wall attachment. Math. Biosci. 158 (2), 95–126. Bocher, B.T.W., Cherukuri, K., Maki, J.S., Johnson, M., Zitomer, D.H., 2015. Relating methanogen community structure and anaerobic digester function. Water Res. 70, 425–435. Castrillón, L., Vázquez, I., Marañon, E., Sastre, H., 2002. Anaerobic thermophilic treatment of cattle manure in UASB reactors. Waste Manage. Res. 20, 350–356. Chandra, R., Vijay, V.K., Subbarao, P.M.V., Khura, T.K., 2012. Production of methane from anaerobic digestion of jatropha and pongamia oil cakes. Appl. Energy 93 (5), 148–159. Cheng, F., Kanokwan, B., Irini, A., 2011. Anaerobic co-digestion of desugared molasses with cow manure; focusing on sodium and potassium inhibition. Bioresour. Technol. 102, 1005–1011. De Vrieze, J., Hennebel, T., Boon, N., 2012. Methanosarcina: the rediscovered methanogen for heavy duty biomethanation. Bioresour. Technol. 112, 1–9. Daniel, I., Massé, Yan, Gilbert, N.M.C., Saady, Charle, Liu, 2013. Low-temperature anaerobic digestion of swine manure in a plug-flow reactor. Environ. Technol. 34 (18), 2617–2624. Dong, L.L., Cao, G.L., Guo, X.Z., Liu, T.S., Wu, J.W., Ren, N.Q., 2019. Efficient biogas production from cattle manure in a plug flow reactor: a large scale long term study. Bioresour. Technol. 278, 450–455. Evans, P.N., Parks, D.H., Chadwick, G.L., Robbins, S.J., Orphan, V.J., Golding, S.D., 2015. Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genomecentric metagenomics. Science 350 (6259), 434. Hao, L.P., Lu, F., He, P.J., Shao, L.M., 2008. Impact of liquid volume of recycled methanogenic effluent on anaerobic hydrolysis. Environ. Sci. 29 (9), 2639–2641. Hu, Y., Shen, F., Yuan, H.R., Zou, D.X., Pang, Y.Z., Liu, Y.P., Zhu, B.N., Chufo, W.A., Jaffar, M., Li, X.J., 2014. Influence of recirculation of liquid fraction of the digestate (LFD) on maize stover anaerobic digestion. Biosyst. Eng. 127, 189–196. Huang, C., Guo, J.H., Wang, C., Xiong, L., Luo, M.T., Chen, X.F., Zhang, H.R., Li, H.L., Chen, X.D., 2015. Efficient continuous biogas production using lignocellulosic
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Reflux of acidizing fluid for enhancing biomethane production from cattle manure in plug flow reactor
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Lili Dong, Guangli Cao , Jiwen Wu, Shanshan Yang, Nanqi Ren State Key Laboratory of Urban Water Resources and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Reflux acidizing fluid Cattle manure Biogas Plug flow reactor Microbial community succession
The performance and microbial community succession of a 36 L working volume plug flow reactor was evaluated for treated cattle manure at an organic loading rate of 2.67 g-TS/L/day, a temperature of 38 °C ± 1 °C, and a hydraulic retention time of 18 days. A reflux of acidizing fluid effectively enhanced anaerobic digestion performance and promoted optimization of microbial community structure. The average biogas volume production rate was 1.08 L biogas/L reactor, which was 116.5% higher than the control without reflux of acidizing fluid. The specific qmethane production yield and methane content reached 0.204 L/g VS and 70%. Moreover, methane yield achieved 0.34 m3/kg removal COD with a COD removal of about 70.56%. The bacteria genera Christensenellaceae, Bacteroidales, vadinBC27, Ruminococcaceae and Treponema_2 were further enriched. Methanosarcina became the dominant methanogen in the whole PFR operation process. This study offers new opportunities for producing renewable energy from enhanced cattle manure biodegradability.
1. Introduction Animal waste is one of the most abundant waste fractions generated worldwide. The amount of manure produced increases daily as the number of livestock and poultry breeding industry grows. Traditionally, manure management has relied on the basic agronomic approach of storage and field spreading for production of forage crops. Although effective for nutrient recycling, these systems can be odor intensive and have adverse environmental impacts in terms of terrestrial eutrophication and greenhouse gas emissions (CHG) (Muhammad et al., 2016; Sommer et al., 2009). Anaerobic digestion (AD) is considered an economical and effective method for the treatment of animal wastes (Cheng et al., 2011; Lansing et al., 2008). In spite of low biogas yields due to its high fiber content, cattle manure is an excellent ‘carrier’ substrate for biogas production in AD of concentrated waste – which would be difficult to treat separately – because of its high water content, high buffering capacity and wide variety of nutrients, which are necessary for optimal bacterial growth (Kaparaju et al., 2009; Angelidaki and Ellegaard, 2003). Various authors have studied techniques to improve manure’s biogas production, including development of low-cost digesters, key operating parameters, and pretreatment technique e.g. (Khalid et al., 2011; Ballyk and Smith, 1999). The sludge refluxing method is an effective control measure mainly used to reduce environmental impact and operate costs and to simplify
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management in urban sewage treatment. Recently, biogas slurry refluxing has been used as a technical method to improve yields in biogas projects (Shahriari et al., 2012; Hao et al., 2008). However, some problems may occur after the long-term operation in industrial application, such as ammonia accumulated, volatile fatty acids (VFAs), nonbiodegradable intermediates, and other inhibitory substances (Hu et al., 2014; Wang et al., 2018). The accumulation could result in changes to the anaerobic environment and inhibition of microbial activity, with the potential to cause complete malfunction of the anaerobic digestion system (Mshandete et al., 2004). Microorganisms are the core of AD, and as this biochemical process involves a great variety of microbial groups, the success of this anaerobic digestion process depends crucially on their growth and metabolism (Bocher et al., 2015; Wang et al., 2018). The plug-flow reactor (PFR) is often referred to as a low-cost anaerobic digester. The contents are not mixed, and continuous management or internal heating is not required when located in a tropical climate (Lansing et al., 2008). The PFR’s separation into acidogenic and methanogenic phases along the flow path of the reactor improves reactor stability and treatment efficiency (Yue et al., 2011; Daniel et al., 2013). As known the limiting step of anaerobic fermentation is hydrolysis, especially for substrates with high lignocellulosic content. So the PFR maybe meet the requirements of staged regulation reactor to improve hydrolysis efficiency. In addition, initial investments for PFR
Corresponding author. E-mail address: [email protected] (G. Cao).
https://doi.org/10.1016/j.biortech.2019.03.092 Received 1 February 2019; Received in revised form 15 March 2019; Accepted 17 March 2019 Available online 19 March 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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investigation.
installation and start-up are typically lower than those for other kinds of reactor configurations because they do not have internal moving parts and usually require less computer control and human intervention. Moreover, a PFR avoids the ‘short-circuit’ phenomenon because it involves the first-in and first-out material and enough hydraulic retention time (Rowse, 2011; Patinvoh et al., 2017; Kothari et al., 2014). However, anaerobic treatment of animal wastes in laboratories, at full scale and under different operating conditions, commonly employs completely stirred tank reactors (CSTR) (Khalid et al., 2011). Although low-cost PFRs have been developed, most of these have focused on design, construction, and optimization. Rowse (2011) has demonstrated a methane yield of cattle manure and straw in a horizontal PFR of 0.163 L CH4/g VS added at a hydraulic retention time (HRT) of 40 d and a temperature of 37 °C. Subsequently, Daniel and Massé et al. (2013) investigated a PFR treatment of pig manure at a low temperature (25 °C) and HRT of 67 d, obtaining specific methane yields ranging from 0.1486 L CH4/g TCOD. However, to the authors’ best knowledge, there is a lack of research on the effect of refluxing acidizing fluid (RAF) on the AD process, especially involving cattle manure as the PFR substrate. Moreover, there have been few studies on the relationship between the transformation process of the substrate and the succession process of microbial communities. The main objective of this paper was to show the profitability and advantages in the design of a PFR with RAF to degrade cattle manure at a lab-scale for 90 days operation. The purpose was to reflect the operation performance of the digester and provide more accurate technical data for guided industrial application. During the digester operation process, 16S rRNA gene sequencing was used to characterize the dynamics of the microbial community structure evolution from operation phase to RAF operation phase. Meanwhile the nutrient of the chemical oxygen demand (COD) removed efficiency, total solid (TS) and volatile solid (VS), pH, biogas production and the content of biogas were determined. This gave a unique opportunity to deep insight the influences of RAF on substrate conversion and microbial community in PFR apparently operating close to the edge of stability.
2.2. Reactor configuration and operation 2.2.1. Reactor configuration A horizontal PFR (Fig. 1) for continuous anaerobic digestion cattle manure process was constructed with transparent Plexiglas sheets with a thickness of ten millimeters in a rectangular shape, which was designed and made at the Harbin Institute of Technology, Harbin. The PFR had a total length of 3 m, which was composed of two 1.5 m reactors in parallel series, and a width of 0.1 m and a height of 0.15 m, 0.03 m for gas space in height (which including the total height), with a capacity of 45 L and an active working volume of 36 L. The PFR was divided into 10 equal compartments, they were K1, K2, K3, K4, K5, K6, K7, K8, K9 and K10 from the inlet to the outlet. The inlet and outlet were on the same side. The reflux port was located in the middle of the K2. The top Plexiglas supported temperature measuring ports, sampling ports and gas collection ports. The external PFR was set up circulating hot water in the space between the reactor Plexiglas walls to ensure the constant temperature required for the digester. 2.2.2. Reactor start-up and operation The PFR was operated with 5% by wt of TS (equivalent to about 50,000 mg/L COD). In order to allow acclimation period for the anaerobic microorganisms within digester, gradual and careful changes in the environment was employed. Stable digester temperature was maintained at 38 ± 1 °C by circulating hot water installation. Prepared substrate was semi-continuously fed into the digester one time a day by pumping the substrate from the homogenizer. Operating pressure was around 1 atm. Pressure from the new feed and biogas inside the reactor pushed out the effluent from liquid surface through the gas collection ports on the top of digester. The outflows were collected in effluent bucket. Biogas from the digester flowed further to gas bags for measuring biogas production and compositions. Biogas production was collected by gas collecting bag from every gas collection hole and measured by dewatering method reversible cycle and registration. The whole fermentation operation process can be summarized into three different phases: start-up (from 1 to 20 day), operation phase (21–40 day) and RAF operation phase (41–90 day). During the RAF operation phase, half of the daily feed amount from the reflux port was added into the substrate before feeding. Experiments were carried out using progressive organic loading rate during start-up stage. The loading rate of 0.67 g-TS/L/day (OLR1) continued only about 2 days. Then it gradually increased to around 1.33 g-TS/L/day (OLR2) in the next 3 days and further increased to 2 g-TS/L/day (OLR3) in the next 6 days. The 2.4 g-TS/L/day (OLR4) continued 5 days. Then the organic load rate was added to the design value 2.67 g-TS/L/day (OLR5) after 16 days and remained at the same level until the end of start-up period and the further operation. From day 21 to operation end, the HRT was 18 days. The inoculum was added at 80% (v/v). Before feeding, an equal amount of digestion was withdrawn.
2. Materials and methods 2.1. Substrate and inoculum characteristics Substrate used in the experiment was cattle manure from WONDERSUN Green Hill Foot Milk Source Base, Harbin. The fresh cow manure was collected in a closed container, stored in the 4 °C for further use and replaced every seven days. The manure was mixed thoroughly and heated 36–40 °C before use. Liquid inoculum used in this research was obtained from a large-scale PFR project of the Modern farming group of the Shuangcheng 3 MW cow manure disposal, which had been running for more than three year under mesophilic conditions (Dong et al., 2019). Table 1 illustrates the most important physical and chemical characteristics of cattle manure and inoculum used in this Table 1 Characteristics of the cattle manure and inoculum. Parameter
Substrate
Inoculum
pH ORP (mv) TS (%) VS (%) TN (mg/L) TP (mg/L) COD (mg/L) VFA (mg/L)
7.76 ± 0.01 −325.00 ± 9.1 15.35 ± 2.35 13.51 ± 0.53 1184.70 ± 25.82 575.21 ± 13.35 159050.00 ± 15.43 1534.60 ± 5.03
7.67 ± 0.01 −221 ± 5.35 3.82 ± 0.01 1.80 ± 0.01 11.37 ± 4.23 85.62 ± 0.46 27975 ± 9.86 234.21 ± 1.45
2.2.3. Analytical methods In the process of starting and stabilizing, inflow and outflow samples were collected from inlet and outlet of the reactor every day. Samples from the K2 and K6 sample holes of the reactor every 5–7 days. Then the pH, oxidation reduction potential (ORP), TS, VS, COD and contents of biogas (CH4 and CO2) were determined every day. The TS and VS were assayed according to Standard Methods (APHA, 1998). The pH and chemical oxygen demand (COD) were detected by HACHHQ40d multi and HACH-DRB 200, respectively. The total nitrogen (TN) and the total phosphorus (TP) were measured by analytikjena multi N/ C 2100s and Perkin Elmer Optima 8300, respectively. The oxidation reduction potential (ORP) was tested by C220-ORP (Shanghai Precision Instruments Co., Ltd). The gas products (CH4 and CO2) were determined by gas chromatography (GC, 7890A, Agilent Cooperation, USA).
VS, volatile solids; TS, total solids; COD, chemical oxygen demand; TN, total nitrogen; TP, total phosphorus; VFA, volatile fatty acids; ORP, oxidation reduction potential. 249
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Fig. 1. Schematic process flow diagram of PFR anaerobic fermentation system.
biogas production, which was only 4 L/d (0.12 L gas/L reactor) with the CH4 content was less than 45% (Fig. 2b). The result may a lag or adaptation period in which the methanogenic bacteria acclimatized to the new environment. Additionally, the accumulated air in the reactor was released by biogas production and emission to lead low CH4 content. In the following days, the biogas production was observed to gradually increase from 5.76 L/d (0.17 L biogas/L reactor) to 21.40 L/d (0.63 L biogas/L reactor) during 4 to 20 d, and the CH4 content maintained a relatively stable value of 56.20% ± 5.13%. This result compared favorably with Jagadish et al. (1998) who reported the biogas production of 0.5 m3 gas/m3 reactor when the digestion of leaf was investigated in PFR, suggesting that the biogas slurry started adapting to the new environment and the digester successfully started up. After successful start-up of the digester, the digester entered operation phase. As shown in the Fig. 2a and b, the daily biogas production fluctuated due to fluctuation of daily OLR, from 14.69 L/d to 22.08 L/d during day 21 to day 40. The average value was 17.16 L per day (0.53 L biogas/L reactor) and the CH4 content maintained a relatively stable value of 57.01% ± 4.94%. This result was similar to or even higher than the average results reported in manure biogas production researches (Wang et al., 2017; Jagadish et al., 1998). The specific biogas yield and methane yield also have a small range of fluctuations, and the average specific biogas and methane yields were 0.15 ± 0.02 L/g VS and 0.10 ± 0.02 L/g VS, respectively (Fig. 2c), which are comparable to those reported by Abubakar and Ismail (2012) and Risberg et al. (2013). Moreover, the biogas production-based removal COD was around 0.39 m3/kg removal COD, with 0.22 m3/kg removal COD CH4 production.
2.2.4. DNA extraction and Illumina sequencing For insighting the change of microbial community succession before/after reflux acidifying liquid, samples (3–10 mL) from the K2 and K6 sample holes were taken over the period of operation phase and after adding reflux acidifying liquid operation phase with a sterilized sample spoon and stored in a 50 mL sterile plastic test tubes at −80 °C before went for DNA analysis. Microbial DNA was extracted from collected samples using the bacterial DNA mini kit (Watson Biotechnologies Co. Ltd., Shanghai, China) according to manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, USA), and DNA quality was checked by 1% agarose gel electrophoresis. Bacterial V3-V4 region of 16S rRNA gene was amplified using the forward and reversed primer 338F (5′- ACTC CTACGGGAGGCAGCAG-3′) and 806R (5′- GGACTACHVGGGTWTCTAAT-3′). Archaea V4-V5 region of 16S rRNA gene was amplified using 524F (5′-TGYCAGCCGCCGCGGTAA-3′). PCR products were purified using GeneAmp (9700 ABI, USA) PCR purification kit (Axygen Biosciences, Union City, CA, USA) and then went for Illumina sequencing platform. The resulted PCR products were quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s protocol. The sequences obtained from Illumina sequencing were analyzed following the pipelines of Quantitative Insights into Microbial Ecology (QIIME) software (www.microbio.me/qiime) as described by the Huang et al. (2015). Taxonomic classification of each phylotype was determined using the SILVA rRNA database project with over 97% of sequence similarity.
3. Results and discussion 3.2. Performance of PFR with RAF 3.1. Start-up and operation of the PFR 3.2.1. Daily biogas production Biogas production is an intuitive indicator for a digester that can reflect the performance of anaerobic digester efficiency and property. To investigate the effect of RAF on the digester operation process, the acidizing fluid from the reflux port was recirculated in the inflow substrate every day from day 41 to the end of operation. The daily biogas production and volume biogas production rate were illustrated
For an anaerobic reactor, a quick and stable start-up is very important. In this study, the start-up was carried out using progressive organic loading rates. Moreover, the inoculum was added at 80% (v/v) to improve the microbial content and shorten the start-up time. It was observed that the daily biogas production increased with the increasing of OLR (Fig. 2a). During the first 3 days of observation, there was less 250
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Fig. 2. Performance of PFR during the whole operation. (a) Daily biogas production and volume biogas production rate; (b) Variation of methane and carbon dioxide content in produced biogas from PFR; (c) Variation of specific biogas (methane) and biogas (methane) production based removal COD; (d) COD removal with different operation phase; (e) pH value of in/out flow and samples of PFR.
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initial days, so the high removal rate of COD was obtained (Fig. 2d). From day 17, the organic load rate was added to the design value 2.67 g-TS/L/day, and the COD removal efficiency retained around 45% during the operation phase. After added RAF, COD removal fluctuated from day 42 to day 72, but overall, COD removal improved. After day 72, COD removal stabilized at about 70.56%, showing that anaerobic fermentation had entered its stationary phase. The fluctuations of COD removal efficiency could be due in part to the substrate of PFR being first in and first out, meaning that the outlet situation of one day was affected by the inlet substrate of the previous 18 days. Also, the cattle manure used in this research was collected every week to ensure that its biodegradable portions weren’t digested due to long-term storage, which meant that input COD fluctuated. Overall, the average COD removal rate increased 25.6% compared with the operation phase, indicating that RAF contributes to the substrate degradation. This also implies that the fermentation system had buffering capacity for unstable inlet COD and thus has potential for industrial application, because unstable COD loading is widespread in actual biogas production on an industrial scale.
(Fig. 2a). It was observed that biogas production significantly increased from day 42. In the following 19 days, daily biogas production continued increase until day 61. After that, biogas production stayed around 37.15 L/d (1.08 L biogas/L reactor) until the digester stopped operation. This result showed a 116.5% increase in biogas production after RAF compared with operation phase. In our previous work, a large-scale PFR project of the Modern farming group of the Shuangcheng 3 MW cow manure disposal, which had been running for more than three year under mesophilic conditions, the volume biogas production rate was 0.58 L biogas/L reactor (Dong et al., 2019). And the operation parameters (refer to fermentation temperature, residence time, reactor type, etc.) of the project were basically the same as this study. Compared with industrial engineering, the result of this study increased 86.21%. This increase was satisfactory, and indicated that the RAF enhancing the raw material biogas production efficiency. 3.2.2. Methane and carbon dioxide content of produced biogas Usually, CH4 and CO2 are the two main components present of biogas. CH4 is the energy product, so CH4 content in biogas is a key index for industrial production of biogas. A higher CH4 content means more valuable biogas production. The content of CH4 in biogas significantly increased during the RAF operation phase (Fig. 2b). The CH4 content was between 45.98% and 69.51% with an average at around 65.2%, more than 8.19% higher than that in the operation phase. Moreover, the maximum CH4 content was close to 70%. This result was similar to that of Yang et al. (2015), who reported that the CH4 content of biogas production from anaerobic digestion was about 53–70%.
3.2.5. pH During AD process, pH is an important parameter affecting the growth of microbes during anaerobic digestion (Yadvika et al., 2004). Inflow and outflow pH were measured every day to evaluate PFR operation process, which partly reflects the conditions of the fermentation system (Fig. 2e). After start-up, the pH of inflow material varied widely from 6.99 to 7.51 between operation phase and RAF operation phase. The main reason was that the pH of the fresh cattle manure collected every week fluctuated greatly. While, there were a small fluctuation for the pH of outflow overall the whole operation process and the pH stable at 7.54 ± 0.12. It indicated that the anaerobic fermentation is always in a normal state after start-up. For the pH change in the digester, K2 and K6 samples of the digester representing the state of the acidogenic and methanogenic phase respectively. It can be seen from Fig. 2e, the pH for the K2 sample reduced significantly when the RAF was added, and it tended to be stable and maintained at around 6.28 after 30 days. This indicates that hydrolytic acidification was increased after RAF. However, the pH of the K6 sample showed small fluctuations (from 7.31 to 7.6) between operation phase and RAF operation phase. According to the report, most anaerobic bacteria, including methane-forming bacteria, perform well within a pH range of 6.8–7.2 (Chandra et al., 2012). In this study, biomethane production remained at a good level at a pH between 6.27 and 7.60. This suggests that the PFR has a wider resistance to the pH of the substrate which would have been very beneficial for anaerobic fermentation to increase biogas production. To summarize, based on system stability, methane production, substrate utilization and pH, RAF could be considered a suitable control measure for enhancing the AD of cattle manure in PFR. In addition, PFR is probably a highly efficient and economic configuration for applying in waste treatment with high lignocellulose content.
3.2.3. Specific biogas and methane production yield Besides biogas production, specific biogas yield (or methane yield) is also an important index in industrial applications (Marimuthu and Kirubakaran, 2015). This index captures the biogas (methane) production per unit VS and per unit removal COD in the case of cattle manure substrate throughout this long 90-day fermentation process (Fig. 2c). During the RAF operation phase, the specific biogas (methane) production yield increased with the fluctuation from day 42. With the RAF continuous operation, the specific biogas (methane) production yield reaching a maximum of 0.354 L/g VS and 0.20 L/g VS on the 59th day which increased nearly 50% than non-RAF. In the following days, the biogas and methane production yield maintained at 0.329 ± 0.016 L/g VS and 0.204 ± 0.015 L/g VS, respectively. Similarly, the yield of biogas production-based removal COD increased significantly as biogas production increased. Although there were large fluctuations, a very satisfactory result was obtained after RAF, the average biogas production-based removal COD was 0.58 m3/kg removal COD and 0.34 m3/kg removal COD CH4 production, which were 48.7% and 54.5% higher than that in the operation phase without RAF. Laboratory-scale studies with upflow anaerobic sludge blanket (UASB) reactor have demonstrated that anaerobic treatment of manure at mesophilic with the production of biogas was 0.20 m3/kg COD removed (Castrillón et al., 2002). Additionally, CSTR, in comparison, produced a biomethane yield of about 0.224 m3/kg TS (Ismail et al., 2012). Meanwhile, the 0.34 m3 CH4 /kg removal COD obtained in this study nearly reached the 0.35 m3 theoretical value. This value is very attractive for industrialization. Thereby, it is very clear that the RAF measure is potentially effective for enhancing biogas and methane production yields from cattle manure waste.
3.3. Structure of microbial community succession A flexible community structure maintains a stable ecosystem function. To better understand the correlation between performance and microbial community structure succession established during the whole fermentation process, bacterial and archaeal communities were analyzed through Illumina sequencing. For stable performance, the microbial community in the reactor should be dynamically balanced. Hence, the samples were taken from K2 and K6 of the 21nd of operation phase and 68th of RAF operation phase. As a result, the samples deviated slightly, but indeed reflected the overall trend.
3.2.4. COD removal COD removal is one important index of reflect the efficiency and stability of fermentation systems. Usually, for an anaerobic fermentation system of cattle manure, COD removal is 51–79% (Rowse, 2011). During the start-up, for the first three days of fermentation, the performance of COD removal was very high, about 90%, but this value gradually decreased to 72.9% in the next days (from 4th to 17th), suggesting that the digestion process properly at the low OLR in the
3.3.1. Comparative analysis of bacterial communities across samples Compared with operation phase, different bacterial community structures were discovered in the RAF operation phase, and their ratio 252
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Fig. 3. Phylum level distribution of bacterial (a) and archaeal (b) community in digeater’s samples at operation phases and RAF operation phase.
This indicated that in the PFR anaerobic digester, most biodegradable substances have completed the process of hydrolytic acidification in the front of the digester, and the back of digester mainly hydrolyzed and acidified substances that are difficult to biodegrade. Xie et al. (2014) reported the genus vadinBC27_wastewater-sludge_group degrades difficult-to-degrade organic substances. It has also been suggested that Ruminofilibacter and Ruminococcaceae play important roles in cellulose hydrolysis in the rumen (Ransom-Jones et al., 2012). According Evans et al. (2015), the Turicibacter group can positively in fluence the host animal by improving the digestion of complex organic matter such as cellulose. Furthermore, recent reports of the 16S rRNA gene and ribosomal intergenic spacer analysis data indicate the presence of Turicibacter bacteria in the rumen and faeces of cattle and pigs, and in rat and goat hindguts (Zhou et al., 2016). A further change in the bacterial community structure was recorded when the RAF was applied to the digester. The differences were as significant as those observed when the substrate was pushed to the outlet. Among Firmicutes, species as Christensenellaceae were predominantly present in the RAF operation phase, 11.17% in K2 and 11.94% in K6. Meanwhile, Bacteroidales, vadinBC27_wastewater-sludge_group, Ruminococcaceae and Treponema_2 were also prominent in K2. However, unlike K2 in the operation phase, the results of the RAF operation phase were different, the dominant bacteria was Ruminofilibacter (16.36%), accompanied mainly with Ruminococcaceae, vadinBC27_wastewater-sludge_group and Treponema_2. Some studies have been conducted on related rapid and highly efficient degradation of matter. We concluded that the Christensenellaceae genera can form a symbiotic association by highly efficiently converting the material of cattle manure, such as cellulose, hemicellulose and lignin, and that the Ruminofilibacter genus was closely related to the high-speed
among the groups varied considerably. From the Fig. 3a, it can be obtained that the Firmicutes and Bacteroidetes decreased in the RAF operation phase. While, the abundance of Spirochaetae increased by nearly 5 times. Additionally, Proteobacteria significantly increased by nearly 50%. Fibrobacteres, as one of the most important functional bacteria for lignocellulose degradation, increased by 13.4 times in the RAF operation phase. Besides, the other functional bacteria such as Synergistetes, Tenericutes and Chloribi also showed obvious increase in the RAF operation phase. The most visual changes to community structure are that several functional bacteria were obviously enriched in the PFR after RAF, while the other bacteria were reduced to a smaller proportion. Based on the biogas production results, it seems that the abundant functional microorganisms and the structures of specific microorganisms of these phyla were more functionally important for improved the digestion of cattle manure wastes in our experiments. Fig. 4a shows the genus classification of the bacterial community at different samples and phases from the PFR. The most dominant bacterial was Bacteroides in the operation phase. Meanwhile, there were other functional bacteria such as Peptostreptococcaceae, Paeniclostridium, Caproiciproducens, Hydrogenispora, Ruminiclostridium, Ruminococcaceae. The Bacteroides form a phylogenetically highly diverse group and are known as hydrolytic fermentative degraders of polymers in mainly anaerobic habitats (Li et al., 2014). Hernon et al. also reported similar findings when performing AD of carbohydrate-rich waste in a mesophilic anaerobic reactor. When the substrate was pushed to the outlet, some of bacteria that contribute to decomposing difficult-to-degrade organic substances appeared, such as Peptostreptococcaceae, Paeniclostridium, Ruminofilibacter, Ruminococcaceae, vadinBC27_wastewater-sludge_group, Turicibacter, norank_f__Draconibacteriaceae and Christensenellaceae, and the contents of these bacteria were no significant difference.
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Fig. 4. Genus level of bacterial (a) and archaeal (b) community in PFR’s samples at operation phases and RAF operation phase: the species, whose sums of percentage in all the samples are less than 1%, are classified as ‘the others’.
digester mainly used H2/CO2 to produce methane, a large amount of volatile acid accumulated. As the substrate was pushed to the outlet, a large amount of Methanosarcina and Methanosaeta was enriched with acetic acid as the methane bacteria metabolic substrate, so the methane bacteria metabolic substrate mainly used acetic acid in the back of the digester. Methanosarcina in AD reactors can be considered beneficial, since this quite robust archaeon is able to use different substrates. Thereby, multiple pathways for methane production because of its relatively high growth rate and ability to tolerate conditions inhibitory to other methanogens, such as the presence of ammonia or volatile acid accumulation (De Vrieze et al., 2012). However, Methanosarcina was interestingly significantly more abundant in the K2 sample of the RAF operation phase (an increase of 46.83%) accompanied with a 46.91% drop in Methanobrevibacter. It is likely that the proportion of Methanosarcina increased significantly after the acidizing fluid flow regulation maybe associate with a higher concentration of the acetic acid reflux group. At the same time, other hydrogenotrophic methanogens: Methanobacterium, Methanocorpusculum and Methanospirillum were enriched. The state of the community structure of K6 after RAF, there no significant change was observed. Therefore, the acetoclastic pathway was dominant in the RAF operation phase. Most interestingly, the total abundance of hydrotrophic methanogens was similar to methamphetamine. Ultimately, under the highly efficient biomethane-producing state of the digester, the microorganisms could achieved a state of balance to make the digester operate efficiently and steadily. We speculated that the RAF promoted the enrichment of Methanosarcina at the front of the digester, which could efficiently and timely consume the hydrolysate, thus improved the overall biogas production efficiency of the digester. To sum up, this research could provide deeper insights into microbial function in the PFR AD process. RAF increased the microorganism in the feed material, then increases the hydrolysis and acidification of the substrate and promotes the growth of methanogens. Ultimately, RAF can realize a balance of microbial hydrolysis and methane production and create high-efficiency biomethane production. The data obtained provide a basis for the further regulation and control of the complex interactions of the bacterial and archaeal communities in industrial full-scale mesophilic biogas plants.
degradation of cellulose. Ruminofilibacter hydrolyzed lignocellulose by secreting a large amount of cellulase and hemicellulase, and produced a lot of acetic acid and butyric acid, which were used by the methanogenic bacteria and the result was in agreement with Patinvoh et al. (2017). This result suggests that the addition of RAF not only increased the characteristic bacteria of hydrolysis acidification in the front of the digester, but also further increased the bacteria that highly efficiently digest difficult-to-degrade cellulose in the back of digester. Meanwhile, based on the results, the digester allowed the coexistence of more bacteria species during the RAF operation phase. In conclusion, the findings clearly demonstrate that in PFR fermentation of cattle manure, the reflux part of acidification liquid of the reactor can promote to increase the microorganism content of feed materials. At the same time, it can enhance the abundance of highly efficient cellulose-degrading bacteria. This is very beneficial to anaerobic fermentation and a main factor for obtaining higher biogas production yield.
3.3.2. Comparative analysis of archaeal communities across samples Understanding the taxonomic distribution and properties of methanogens allowed us to infer methanogenic pathway at different parts and phases of the digester. Euryarchaeota and Bathyarchaeota were the dominant archaea phyla in the operation phase and RAF operation phase (Fig. 3b). However, there were differences in these two operation phases. Euryarchaeota in the RAF operation phase was 15.44% higher than in the operation phase. On the contrary, Bathyarchaeota was much less present in the RAF operation phase. Additionally, a small amount of WAS2 presented after added RAF. From the results of archaea phyla, the RAF measure can promote the adjustment of the population structure of archaea in the system. Genus-level classification was conducted to further determine phylogenetic discrimination and possible functions of the archaeal members in the digester during different phases. The predominant genera were Methanobrevibacter (72.11%) and Methanocorpusculum (15.68%) in the K2 sample of the operation phase (Fig. 4b). Methanobrevibacter and Methanocorpusculum can use H2 /CO2 as a substrate of methanogenesis. Methanocorpusculum can also use parts of formic acid produced by methane. This suggests that methane mainly produced the H2 reduction of CO2 in the front of digester. In K6 of the operation phase, the dominant archaea were Methanosarcina with relative abundance of 35.12% and Methanobrevibacter with 23.20%. Also present were norank_p__Bathyarchaeota with 17.35%, Methanosaeta with 9.38% and Methanocorpusculum with 9.16%. Because the methanogens at the front of
4. Conclusions This study investigated RAF as one efficient control measure technology for enhancing biogas production and optimizing the microbial 254
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community structure in a PFR digester. Peak daily biogas production reached 39.32 L/day with 1.14 L biogas/L reactor and 0.329 ± 0.016 L/g VS with a CH4 content of close to 70% and a COD removal rate of 70.56% in the PFR during the RAF operation phase. Meanwhile, 0.34 m3/kg removal COD CH4 production was achieved. Many functional microorganisms enriched hydrolytic acidification, especially bacteria that digest difficult-to-degrade cellulose-for example, Christensenellaceae, Ruminofilibacter and Fibrobacteres. Methanosarcina was dominant during the RAF operation phase. Therefore, this technology has practical guiding significance for industrial applications.
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