Anaerobic thermophilic digestion of Maotai-flavor distiller's grains

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Biofuels and Biomass

Anaerobic thermophilic digestion of Maotai-flavor distiller’s grains: Process performance and microbial community dynamics Tianjie Ao, Ruiling Li, Yichao Chen, Chang Li, Zhidong Li, Xiaofeng Liu, Yi Ran, and Dong Li Energy Fuels, Just Accepted Manuscript • Publication Date (Web): 04 Sep 2019 Downloaded from pubs.acs.org on September 4, 2019

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Anaerobic thermophilic digestion of Maotai-flavor distiller’s grains: Process performance and microbial community dynamics Tianjie Ao†, §, Ruiling Li†, ∥, Yichao Chen†, Chang Li†, Zhidong Li†, Xiaofeng Liu†, Yi Ran *, ‡,

Dong Li *, †

†Key

Laboratory of Environmental and Applied Microbiology, Environmental Microbiology

Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Science, Chengdu 610041, China; ‡Biogas

Institute of Ministry of Agriculture, Chengdu 610041, China

§University

∥College

of Chinese Academy of Sciences, Beijing 100049, China;

of Engineering, Northeast Agricultural University, No.600, Changjiang Road,

Xiangfang District, Harbin, Heilongjiang 150030, P.R.China;

ACS Paragon Plus Environment

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KEYWARDS: Maotai-flavor distiller’s grains; Anaerobic thermophilic digestion; Process performance; stability; Microbial community dynamics.

ABSTRACT

Improperly disposed Maotai-flavor distiller’s grains (MDG) cause environment pollution. However, MDG’s high organic matter and moisture contents make this byproduct suitable for anaerobic digestion. The performance and stability of thermophilic anaerobic digestion were evaluated at an organic loading rate of 3 g VS/(L·d) and a working volume of 55 L. The microbial community dynamics were analyzed by highthroughput Illumina pyrosequencing. The volumetric biogas production rate of 1.30 L/(L·d) was obtained upon reaching stability by diluting feed without effluent recirculation.

Defluviitoga,

Candidatus

Caldatribacterium,

Hydrogenispora,

Acetomicrobium,

Methanosarcina, and Methanothermobacter were the dominant acidogenic and


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methanogenic genera. Recirculating the effluent without dilution caused acidification and instability due to its high levels of lactic (74 g/kg), acetic (31 g/kg), and succinic (24 g/kg) acids originally present in MDG. Once acidified, the digester failed to recover using control measures such as halting effluent recirculation, adding trace elements, stopping feeding, and adding inoculum.


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1. INTRODUCTION Maotai-flavor liquor is the national liquor of China. Its popularity has resulted in excessive accumulation of the distiller’s grains, which is the mainly solid byproduct of the brewing process. Now an urgent environmental concern, Maotai-flavor distiller’s grains (MDG) were traditionally landfilled, causing undesirable odors, soil pollution, and land devaluation. This type of treatment has resulted in the loss of a high value-added material and severe environment contamination. Hence, effective and environmentally friendly disposal methods for MDG are needed. The main components of MDG are the distilled sorghum and wheat, which containing high amounts of organic matter. These distiller’s grains may be utilized as feed, fertilizer, and a source of energy. Buttrey, et al. 1 proposed using distiller’s grains as feed for the beef industry. Fruet, et al.

2

reported that corn distiller’s grains are ordinarily used as energy and protein sources for

finishing cattle. However, there are large differences between different kinds of distiller’s grains. MDG contains a high content of rice husk (16.6%) and has a low pH value (3.76) (Table 1), which make it inappropriate for animal feed utilization. Composting has been proposed as an effective process for the valorization of organic waste to produce a stable and nutrient-rich bio-fertilizer.3 However, energy and nutrition elemental (C, H, and N) losses are severe during high-temperature aerobic composting. Distiller’s grains may also be used for energy recovery, such as in thermochemical conversion (e.g. combustion, pyrolysis, and gasification) and biological treatment (e.g. anaerobic digestion (AD)). Thermochemical conversion is widely used for dry hydrocarbonbased materials to recover energy and produce value-added byproducts.4 However, the high moisture content (51.85%) of MDG makes it unsuitable for thermochemical conversion (Table 1). Moreover, MDG’s high N content (C/N=11.45) will also lead to the formation of NOX after the


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thermochemical conversion, resulting in air pollution and nitrogen loss.4 In comparison, the operation of an AD system wastes little energy and element and causes little environmental disturbance. On the contrary, AD can effectively degrade most kinds of organic wastes and simultaneously produce biogas and bio-fertilizer. Hence, AD is a more feasible treatment method for MDG and was thus selected to help solve the environmental issues mentioned above. Various organic wastes had been thoroughly investigated to evaluate the performance and stability of an AD system. Li, et al. 5 researched the instability during thermophilic digestion of vegetable waste, and found that the stable operational condition was maintained at an organic loading rate (OLR) of 1 g VS/(L·d). Zhang, et al. 6 investigated the AD performance of food waste by optimizing mixing strategy, which gained the optimal specific methane yield of 437 mL CH4/g VS under the OLR of 2.4 g VS/( L·d). Gou, et al. 7 conducted the anaerobic co-digestion of waste activated sludge and food waste to evaluate the performance under different OLR, which obtained the best bearing capacity at extremely high OLR of 7 g VS/( L·d). However, the performance of AD on MDG has not yet been investigated. Exploring the performance and stability of an AD system treating MDG is indispensable to provide guidelines for engineering applications. In this study, the feasibility of AD of MDG was evaluated. Various gaseous (biogas contents) and liquid (ammonia nitrogen, alkalinity and volatile fatty acids (VFAs)) parameters were monitored daily. Different measures were adopted to control AD system including: without effluent recirculation (i.e. water dilution of feed), effluent recirculation, the addition of trace elements (TEs), and the addition of inoculum. These measures were applied to evaluate the performance and stability of thermophilic digestion of MDG under a certain OLR. The microbial community structure and dynamics under different conditions were monitored using 16S rDNA sequencing.


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2. MATERIALS AND METHODS 2.1 Feedstock and inoculum MDG was acquired from the Kweichow Moutai Group in Moutai town, Renhuai city, China, and stored in a refrigerator at 4 oC before use. The characteristics of MDG are list in Table 1. The inoculum came from a thermophilic anaerobic digester fed with vegetable wastes and pig manure. It was acclimated for 30 days by feeding MDG at 55 oC, until the methane content in the biogas was above 60%. The pH value of the inoculum was 8.28, and the proportion of total solid (TS) and volatile solid (VS) in the inoculum were 1.32% and 0.87%, respectively. Table 1 Characteristics of Maotai-flavor distiller’s grains Parameters

Value

Parameters

Value

pH

3.76

Succinic acid (g/Kg)

24.12

Total solid (TS) (g/Kg)

481.50

Lactic acid (g/Kg)

74.16

Volatile solid (VS) (% TS)

92.19

Acetic acid (g/Kg)

30.66

Crude protein (% TS)

23.42

Formic acid (g/Kg)

0.18

Crude lipid (% TS)

6.00

Levulinic acid (g/Kg)

4.62

Cellulose (% TS)

9.70

N (%)

3.78

Hemicellulose (% TS)

2.60

C (%)

43.38

Lignin (% TS)

16.60

H (%)

6.54

Rice husk content (%TS)

16.60

C/N

11.45


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Figure 1. Experimental setup

2.2 Experimental setup and design The experimental setup included an anaerobic reactor, an online gas monitoring component and a liquid monitoring component (Figure 1). The total volume of the reactor was 70 L, with a working volume of 55 L, accounting for the blocking of the gas pipe and gas leakage from feeding pipe. The reactor was operated under thermophilic conditions (55 ± 2 oC). The content in the reactor was mixed intermittently 8 times per day at 30 rpm for 30 min. MDG was mixed with tap water or effluent at a specified proportion and fed into the reactor every day. The designed OLR and hydraulic retention time (HRT) were fixed at 3 g VS/(L ·d) and 20 d by controlling the total feed to 2750 g. Table 2 lists the daily feed by manual work under different control measures. At the control measure of without effluent recirculation by adding water (A), the effluent was discharged in a total amount of 2750 g before feed. And the total amount of feed was 2750 g that contained MDG of 371.6 g and water of 2378.4 g. At the control measure of effluent recirculation (B), the effluent was discharged in a total amount of 2750 g, subsequently, it sieved through a 0.6


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mm mesh to filter out solid digestate. And the total amount of feed was 2750 g that contained MDG of 371.6 g and filtered liquid effluent of 2378.4 g. The trace elements were applied following quantities reported by Li, et al. 8 ( i.e. 1.0 mg/(L·d) Fe, 0.1 mg/(L·d) Co, and 0.2 mg/(L·d) Ni) under the condition of C during days 58-77. No feeding and no discharging were applied during days 84-95 (stopping feeding, D). At the control measure of dilution by adding water into the reactor (E), the effluent of 2750 g was discharged before the same amount of water was fed. At the control measure of adding inoculum (F), the same amount of inoculum was added into the reactor after the effluent of 2750 g was discharged from the reactor. Table 2 Operating conditions for the thermophilic digestion of Maotai-flavor distiller’s grains Running

OLR

HRT

Daily feed (g)

Daily

Control

(g VS/L·d)

(d)

MDG

Water

effluent (g)

measure

1-40

3

20

371.6

2378.4

0

0

2750

A1

41-57

3

20

371.6

0

2378.4

0

2750

B1

58-77

3

20

371.6

0

2378.4

0

2750

C1

78-83

3

20

371.6

2378.4

0

0

2750

A1

84-95

0

/2

0

0

0

0

0

D1

96-102

0

20

0

2750

0

0

2750

E1

103-108

0

20

0

0

0

2750

2750

F1

109-145

3

20

371.6

2378.4

0

0

2750

A1

time (d) Effluent

Inoculum


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Notes: 1A: without effluent recirculation by adding water, B: Effluent recirculation, C: Effluent recirculation + Trace elements, D: Stopping feeding, E: Dilution by adding water into the reactor, F: Adding inoculum. 2The HRT was meaningless under the condition of D without any feeding.


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2.3 Analytical methods TS and VS were measured using standard methods.9 The proportions of C, H, N were measured by a Vario EL element analyzer (Elementar Analysensysteme GmbH, Germany). The Van Soest chemical titration method was used to measure the contents of hemicellulose, cellulose, and lignin in the MDG.10 The compositions of crude lipid and crude protein were determined based on Chinese standard methods (GB/T 5009–2003). The contents of succinic, lactic, formic, and levulinic acids were quantified using a high-performance liquid chromatograph (HPLC, SHIMADZU LC-20 AD, Japan) equipped with an Aminex HPX-87 column (Bio-Rad), a RID10A refractive index detector and an SPD-20 UV/vis detector.11 Biogas production was measured with a gas flowmeter (Beijing Sevenstar Electronics Co., Ltd, China). Biogas contents were analyzed using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a thermal conductivity detector and a 3-m stainless steel column packed with MOLSIEVE 13X (80/100 SS).12 The pH and ORP values were monitored online every day. The digestate was sampled and sieved through a 0.6 mm mesh to filter out large insoluble particles before feeding. The filtered liquid samples were centrifuged twice at 12000 rpm for 5 min and subsequently passed through a 0.45 μm filter to measure the contents of ammonia nitrogen, alkalinity and volatile fatty acids (VFAs). Nessler’s reagent spectrophotometry was conducted to determine the ammonia nitrogen using a TU-1810UV-VIS spectrophotometer (Beijing Persee Instrument Corp., China). Alkalinity was measured using a ZDJ-4B automatic potentiometric titrator (Shanghai Precision & Scientific Instrument Co., Ltd., China). Based on a previously published method13, the pH endpoints of bicarbonate alkalinity (BA), intermediate alkalinity (IA) and total alkalinity (TA) were 5.75, 4.3 and 3.7, respectively. VFAs were measured with a gas chromatograph equipped with a flame ionization detector and a 30 m × 0.25 mm × 0.25


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mm capillary column (DB-FFAP, USA).12 2.4 DNA extraction, PCR amplification, and high-throughput Illumina pyrosequencing The digestate was sampled at specific days during the stable stage (days 19, 25, 31, 34, 40), the first unstable stage (days 52, 55, 58), the recovery stage (days 61, 64, 67), the second unstable stage (days 70, 73, 76), the second recovery stage (days 97, 100, 103) and the collapsed stage (days 141, 144, 145). These stages were defined at the result and discussion part. All samples were centrifuged at 5000 rpm for 10 min, then the pellets were frozen immediately at -80 oC. Microbial DNA was extracted using the E.Z.N.A.

®

soil DNA Kit (Omega Bio-tek, Norcross, GA, USA)

according to the manufacturer’s protocols. PCR was performed with the TransStart Fastpfu DNA Polymerase (TransGen Biotech, Beijing, China) using the 515F and 806R universal 16S rRNA gene primers.14 Purified amplicons were pooled in equimolar concentrations and paired-end sequenced (2× 300 bp) on an Illumina MiSeq platform (Illumina, San Diego, USA) by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The Majorbio I-Sanger Cloud Platform (www.i-sanger.com), an online tool, was used to analyze the structure and distribution of the microbial consortium at each stage. 3. RESULTS AND DISCUSSION 3.1 Characteristics of Maotai-flavor distiller’s grains The main characteristics of MDG are listed in Table 1. Due to a high amount of total solid (481.5 g/kg), the feed concentration was adjusted by either effluent recirculation or by water dilution. Yadav, et al.

15

revealed the role volatile solid (VS) play in determining the organic

content of the substrate. In our previous study, we clarified the specific definitions of biodegradable VS and refractory VS.16 The VS content in dry MDG is 92.19%, while the refractory VS (rice husk) accounts for 16.60% of dry MDG. This result indicates that biodegradable forms


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of organic matter (such as lactic acid, succinic acid, VFAs, starch, lipid, and protein) are dominant in MDG, which make it suitable for AD. Overall, anaerobic digestion is a feasible treatment for MDG, decreasing environmental pollution and costs while increasing cleaner energy production. However, the high contents of organic acids (lactic acid, acetic acid and succinic acid) and low C/N in MDG may result in acidification and ammonia inhibition, especially if effluent is recirculated without adding water for dilution. 3.2 Process performance and stability 3.2.1 Biogas production and components Biogas production is one of the most important parameters used to evaluate the stability and dynamics of anaerobic systems. Biogas production is commonly expressed as volumetric biogas production rate (VBPR). The utilization of raw material can be expressed in terms of specific biogas production rate (SBPR). The VBPR, SBPR, and biogas contents from a single run are shown in Figure 2. The control measure of without effluent recirculation by adding water (A) was conducted at the OLR of 3 g VS/(L·d) for 40 days. During this period, the VBPR, SBPR and methane content were stable at 1.30 L/(L·d), 0.43 L/(gVS·d), and 60.33%, respectively. Thus, this period was defined as stable stage (Ta). At Ta stage, an OLR of 3 g VS/(L·d) results in stable performance when feed is diluted by adding water.


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Figure 2. The daily changes of (a): biogas contents, (b): volumetric biogas production rate (VBPR)


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and (c): specific biogas production rate (SBPR). A: Without effluent recirculation by adding water, B: Effluent recirculation, C: Effluent recirculation + Trace elements, D: Stopping feeding, E: Dilution by adding water to the reactor, F: Adding inoculum. Ta: Stable stage, Tb: First unstable stage, Tc: Recovery stage, Td: Second unstable stage, Te: Second recovery stage, Tf: Collapsed stage.

Vuitik, et al. 17 reported that the effects of effluent recirculation on AD performance depend on the configuration of the reactor and the characteristics of the raw materials. To reduce water use, prevent the loss of microorganisms, and improve the utilization of raw materials, we changed the control measure to effluent recirculation (B) from day 41 to day 57. During this period, the VBPR remained stable for the first 10 days, and then it began to continuously decline from 1.45 L/(L·d) to 0.96 L/(L·d). The same trends were observed for SBP and methane content. The SBP dropped from 0.47 L/(gVS·d) to 0.32 L/(gVS·d) and the methane content decreased from 61.12% to 51.78%. We designated this period as first unstable stage (Tb). These results indicate that effluent recirculation does not enhance the performance of AD treating MDG; in fact, this measure diminishes AD performance, and this may be due to the inhibitory effect of organic acids or ammonia. To prevent further instability, the control measure was changed to effluent recirculation + trace elements (C) from day 58 to day 77. The VBPR, SBP and methane content gradually recovered to 1.61 L/(L·d), 0.53 L/(gVS·d), and 59.67%, respectively, from day 58 to day 68. Unexpectedly, these parameters sharply decreased to 0.73 L/(L·d), 0.19 L/(gVS·d), 47.5%, respectively, from day 69 to day 77. The first 11 days and the last 9 days of control measure C were defined as the recovery stage (Tc) and second unstable stage (Td), respectively.


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A series of control measures were conducted to prevent the further instability from day 78 to day 108. These measures consisted of the following: without effluent recirculation by adding water (A), stopping feeding (D), dilution by adding water to the reactor (E) and adding inoculum (F). The VBPR continuously declined to 0.14 L/(L·d) during this whole period. However, the methane content increased slightly to 59.24% from day 78 to day 103 under control measures A, D, and E. Control measure F increased the carbon dioxide content, which could be explained by the acidogenesis from the added inoculum exceeding methanogenesis. Therefore, this whole period was defined as second recovery stage (Te). After the process recovered, the system was returned to the OLR of 3 g VS/(L·d) without effluent recirculation (A) from day 108 to day 145. The VBPR sharply increased to 1.20 L/(L·d) and maintained this level for 12 days. The methane content and the SBPR followed the same trend. Surprisingly, the VBPR, SBPR and methane content suddenly decreased from day 122 to day 145 until biogas production was negligible. The period from day 108 to day 145 was thus defined as collapsed stage (Tf). Although the same operational conditions were adopted in stages Ta and Tf, the biogas production levels totally differed, a difference that can be explained by microbial analysis.


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Figure 3. The daily changes of (a): pH and oxidation-reduction potential (ORP), (b): Total ammonia nitrogen (TAN), (c): Alkalinity.


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3.2.2 Ammonia, pH, ORP, and alkalinity Online monitoring of pH is relatively easy and convenient, although pH changes cannot reflect the real-time status of AD system with a strong buffering capability.18 ORP is also monitored online and Li, et al.

19

reported that certain ORP trends are highly relevant to the

overloading of an AD system. In this study, pH and ORP were negatively correlated (Figure 3a). Three sudden drops of pH values to 6.8, 5.96 and 5.22 were observed at the Tb, Td and Tf stages, respectively. These pH values are beyond the suitable pH range of 6.8-7.2.20 The pH continued to decrease even when control measure A was implemented during stage Td. However, implementing control measure D successfully stopped the decrease in pH. Measures E and F were subsequently implemented stepwise to further increase pH to a suitable level of 7.4. At stage Tf, the pH value stabilized for 15 days, and then sharply dropped to 5.22. The reactor pH depends on total ammonia nitrogen (TAN), alkalinity, and VFA buffers. TA consists of BA (HCO3-) and IA (VFA-). Fluctuations in TAN, TA, BA and IA were determined by the control measures being implemented (Figure 3b, c). Normally, TAN and TA share similar trends. During stage Ta without effluent recirculation, both TAN and TA tended to decrease due to their dilution. After effluent recirculation, both TAN and TA tended to increase. Either BA or IA may indirectly mirror the accumulation of VFAs in an AD system.19 The fluctuations in BA and IA are presented in the Figure 3c. The tendency of BA and IA had negative relevance. IA tended to increase during the first two unstable stages, while it tended to decrease during the two recovery stages. IA was maintained at a stable level, while BA decreased to zero at stage Tf. This result is highly relevant to the fluctuations observed for VFAs, which demonstrate the instability of AD system, which is directly caused by the accumulation of VFAs.


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Figure 4. (a), (b), (c), (d): The daily changes of individual volatile fatty acids. (e): The changes of lactic acid concentration under the control measures of without effluent recirculation (A) and effluent recirculation (B).

3.2.3 Volatile fatty acids The concentrations of individual VFAs are illustrated in Figure 4. The concentrations of


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VFAs are regarded as the most essential parameter for evaluating the stability of AD systems.21 VFAs are the key intermediates produced from acidogenesis and that function as substrates for methanogenesis.20 The accumulation of VFAs indicates an imbalance between acidogenesis and methanogenesis, which can directly cause the failure of an AD system.5 In this study, acetate, which was the only volatile fatty acid detected in stage Ta, was maintained at a concentration of 614.77 mg/L. During stage Tb, the concentrations of acetate, propionate, n-butyrate, iso-butyrate, n-valerate, and iso-valerate increased to 3102.17 mg/L, 2521.25 mg/L, 80.98 mg/L, 223.53 mg/L, 61.12 mg/L, and 441.89 mg/L, respectively, after effluent recirculation. Siegert and Banks

22

reported that AD systems face imminent collapse when the total VFA concertation reaches beyond 4000 mg/L. Furthermore, the digester failure will occur if the accumulated propionate concertation exceeds 3000 mg/L.20 Thus, the control measure implementing effluent recirculation is likely to result in acidification of AD treating MDG under an OLR of 3 g VS/(L·d). Numerous studies point out that effluent recirculation is beneficial to the AD system because it can prevent losses of alkalinity and microorganisms.23, 24 However, the effluent recirculation adopted in this study negatively affected the AD system. According to Table 1, MDG contains high concentrations of lactic acid (74.16 g/Kg), acetic acid (30.66 g/Kg), and succinic acid (24.12 g/Kg). Therefore, feeding undiluted MDG into the reactor and then implementing effluent recirculation is likely to result in the accumulation of these existing organic acid. The accumulated organic acids inhibit methanogens, which result in the imbalance between the acidogenesis and methanogenesis.25 When without effluent recirculation was adopted, MDG was diluted with water before feeding into the reactor, resulting in the dilution of organic acids, which no longer inhibited methanogenesis. This reasoning is confirmed by Figure 4e, which compares the lactic acid concentration in the stable stage without effluent recirculation (A) to that of the dramatically


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increased lactic acid concentration in the first unstable stage with effluent recirculation (B). Trace elements were added into the reactor at stages Tc and Td, in an attempt to recover the acidified system caused by effluent recirculation. Trace elements are indispensable for the survival and metabolism of methanogens.26 For example, they constitute part of the metalloenzymes involved in the main biochemical pathways of methanogenesis. Hence, the supplementation of trace elements into the AD system was expected to enhance methanogenesis and help recover the unstable system. The addition of trace elements succeeded in improving methane production; it also increased reactor pH, and reduced acetate concentration at stage Tc. However, it did not control the rise of other VFAs. At stage Td, the acetate concentration rapidly increased to 4289.04 mg/L, with other VFAs also trending upward. This result indicates that despite the positive effect of trace elements, they were unable to overcome the negative effect of the continuous accumulation of organic acids when effluent was recirculated. All VFAs stopped increasing, but they did not decrease significantly after control measure A was implemented at stage Te. Therefore, control measure D (no feeding) was implemented to reduce VFA concentrations. However, the VFAs remained stable at high concentrations without any decrease. Subsequently, control measures E and F were implemented, resulting in a remarkable decrease in VFAs, reaching a low level at the end of stage Te. At this point, the system was considered to have recovered, and feeding was re-started with an OLR of 3 g VS/(L·d) at day 108. The system remained stable during the first 10 days of stage Tf. However, reactor stability during stage Tf was less than that in stage Ta in terms of the trend fluctuations presented in Figure 4. The temporary stability finally collapsed with steep increases in the levels of acetate, n-butyrate, n-valerate. Although the same conditions were in operation during stages Ta and Tf, differences


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in VFA concentrations were significant. The collapse of the AD system was likely caused by large changes in microbial community structure and the microbial community at stage Tf was more vulnerable than that of stage Ta.


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Figure 5. (a): The community abundance and dynamics of microbial taxonomic groups in the thermophilic (55 ℃) condition at different performance stages. (b): The relative abundance of dominant methanogens. (c): The dominant Hydrogen-producing and acid-producing bacteria.


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3.3 Microbial community dynamics 3.3.1 Dynamics of the microbial community The major microbial genera with relative abundances greater than 1% are shown in Figure 5. The relative abundance of Defluviitoga tended to remain stable at 34% during stages Ta, Tb, Tc and Td, but sharply decreased to 4% and 1.9% at stages Te and Tf, respectively. The relative abundance of Candidatus Caldatribacterium gradually increased from 5.9% at stage Ta to 24% at stage Tc, then decreased to 0.1% at stage Tf. The relative abundance of Acetomicrobium tended to increase from 2.5% at stage Ta to 17.8% at stage Te, and then decreased to 4.5% at stage Tf. The relative abundance of Hydrogenispora decreased from 14.3% at stage Ta to 7% at stage Tb, and then maintained an average level of 9% at stages Tb, Tc and Td. Finally, it decreased to 5% at stage Tf. The relative abundance of Methanothermobacter and Methanosarcina increased from 2% and 0.3%, respectively, at stage Ta to 8% and 2% at stage Tb, respectively. Subsequently, both genera gradually decreased to 0.2% and 0, respectively, at stage Tf. Defluviitoga has been reported to be abundant in a thermophilic reactor, where it can degrade amino acids and organic acids to form acetate, hydrogen, and carbon dioxide.27, 28 Hydrogenispora, Acetomicrobium, and Candidatus Caldatribacterium have also been reported to function in hydrogen and acid production in the AD system.29,

30

In this study, these acidogens were the

dominant genera during the first four stage (Ta, Tb, Tc, and Td), indicating that acidogenesis continued to be active during these stages. Among the archaea, Methanothermobacter functions as a hydrogenotrophic methanogen, while Methanosarcina functions as a mixotrophic (acetoclastic, hydrogenotrophic, and methylotrophic) methanogen.28,

31

The proportion of

Methanosarcina was much lower than that of Methanothermobacter during the overall process, indicating that the dominant methanogenic reaction performed by Methanothermobacter consisted


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of H2 and CO2 consumption to produce CH4. During the anaerobic co-digestion for fats, oil, and grease with municipal sludge, the dominant methanogens were Methanosaeta and Methanospirillum.32 Sun, et al. 33 investigated the microbial community during continuous AD of straw and cow manure, and found that the prominent methanogens were Methanobrevibacter in the cow manure samples and Methanosarcina in the straw and cow manure mixture. The dominant methanogens found in the different raw materials and reactors in these previous studies obviously differ from the methanogens found in this study. Caproiciproducens (27.4%) and Coprothermobacter (9.3%) were the dominant genera during the collapsed stage (Tf), which differed completely from the dominant genera during the previous stage. Caproiciproducens produces H2, CO2, ethanol, acetic acid, butyric acid, and caproic acid as metabolic end products from anaerobic galactitol fermentation.34 Coprothermobacter is a syntrophic, proteolytic bacterium.35 The system was stable during stage Ta. however, the relative abundance of methanogens during this stage was low. This may be due to the slow growth of methanogens and the loss of methanogens by dilution. Methanothermobacter and Methanosarcina began to increase when the effluent recirculation was implemented (Tb) and began to decrease when continuous effluent recirculation was implemented (stages Tc). During the Td stage, the relative abundance of acidproducing bacteria (Defluviitoga, Acetomicrobium, and Coprothermobacter) continuously increased and the relative abundance of methanogens (Methanothermobacter and Methanosarcina) kept decreasing, which resulted in the maximum accumulation of total VFAs over 10, 000 mg/L. Even the addition of trace elements did not prevent the reduction of methanogens. However, acidogens were maintained at a relatively high level during effluent recirculation. Therefore, effluent recirculation caused the imbalance between acidogenesis and methanogenesis. The


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microbial community changed significantly at stage Te after control measures A, D, E, and F were implemented. Based on the decreased levels of VFAs seen in Figure 4, the system appeared to have recovered. However, considering the microbial dynamics, the system had not really recovered at stage Te. The decrease in VFA levels likely resulted from dilution rather than consumption by methanogens. The methanogens and acidogens (including Defluviitoga, Hydrogenispora, Acetomicrobium, and Candidatus Caldatribacterium) all decreased sharply at the collapse stage (Tf). At this time, Caproiciproducens became the dominant genus, indicating that Caproiciproducens rather than the other four acidogens was more favored to grow under an acidified environment. Generally, the fermentative pathway changes from acid production to ethanol production in order to avoid further acidification of the environment. Caproiciproducens has been reported to carry out ethanol production and is likely to have engaged this fermentative pathway during stage Tf.

Stages

Alpha diversity index

Table

Simpson

Chao

Simpson evenness

Ta

0.174

174.001

0.049

Tb

0.231

159.141

0.039

Tc

0.212

140.640

0.041

Td

0.197

151.037

0.047

Te

0.116

193.905

0.054

Tf

0.096

250.316

0.053

3

Summary of alpha diversity index


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3.3.2 Relationship of the overall microbial community The α-diversity indexes during the different stages are listed in Table 3. The microbial community diversity is reflected by the Simpson diversity index. The microbial abundance and the Simpson diversity index values are negatively related. Microbial diversity decreased during stage Tb (relative to stage Ta), and diversity gradually increased during stages Tc, Td, Te and Tf. These results indicate that effluent recirculation (Tb) reduced microbial diversity, and the subsequent control measures consisting of effluent recirculation + trace elements (Tc and Td), no feeding and no discharging, dilution by water, and adding inoculum (Te) increased the overall microbial diversity. The highest level of microbial diversity was found in the collapsed stage (Tf), indicating that the functional microorganisms were not dominant in the collapsed AD system. The Chao index was used to evaluate microbial richness, and Chao index values increased along with the richer microbial species. The microbial richness decreased during stages Tb and Tc, and increased during stages Td, Te and Tf. The Simpson evenness index, which reflects the evenness of microbial community, decreased at stage Tb, and gradually increased at stages Tc, Td, Te, and Tf. To clarify the effect of various control measures on the microbial distribution, β-diversity was analyzed by principal co-ordinates analysis (PCoA) (Figure 6). Clusters Ⅰ, Ⅱ, Ⅲ, and Ⅳ are located in different areas of the plot, which indicates big differences between each other. The difference between clusters Ⅰ and Ⅱ indicates that effluent recirculation can cause great changes in the microbial community. Groups Tb, Tc, and Td cluster closely together in cluster Ⅱ, which suggests that the addition of trace elements caused no significant difference in microbial distribution. The distance between clusters Ⅰ and Ⅱ is much closer than that between clusters Ⅱ and Ⅲ, suggesting that the subsequent control measure of adding inoculum at stage Te changed


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the microbial distribution of AD system. The red arrow in the figure reflects the overall tendency of microbial

Figure 6. Differences of microbial communities revealed by principal co-ordinates analysis (PCoA).

community dynamics in the AD system over time. The microbial community structure during process failure is represented by cluster Ⅳ, which is totally different from that during the first stable stage (cluster Ⅰ). This result provides the reason why the reactor failed even after the operational conditions were changed back to the initial control measure of without effluent recirculation at an OLR of 3 g VS/(L·d).


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CONCLUSIONS Stable thermophilic digestion of MDG was obtained at an OLR of 3 g VS/(L·d) using a diluted feed; this resulted in a VBPR of 1.30 L/(L·d) and SBPR of 0.43 L/(gVS·d). Effluent recirculation without dilution caused acidification and instability due to the high content of lactic, acetic, and succinic acids in MDG. The acidified AD system remained irrecoverable through a series of control measures. Defluviitoga, Hydrogenispora, Acetomicrobium, Candidatus Caldatribacterium, Methanothermobacter, Methanosarcina were the dominant acidogenic and methanogenic microbial genera in the stable system. Effluent recirculation and adding inoculum disturbed the microbial structure remarkably, however, adding TEs had no significant effect. CORRESPONDING AUTHOR *Corresponding author

Address: No. 13 Section 4, Renmin Nan Road, Chengdu, Sichuan, P.R. China Tel.: +86 2885230679 Fax: +86 2885230679 E-mail address: [email protected]

Address: No. 9 Section 4, Renmin Nan Road, Chengdu, Sichuan, P.R. China Tel.: +86 2882890229


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Fax: +86 2882890229 E-mail address: [email protected]

NOTES Disclaimer: The statements made herein are solely the responsibility of the authors. The authors declare no competing financial interest. ACKNOWLEDGEMENTS This research was jointly supported by the National Key R & D Program of China (2018YFD0501405), Chengdu International Science and Technology Cooperation Project （ 2019-GH02-00024-HZ ） , West Light Foundation of Chinese Academy of Sciences (No. 2018XBZG_XBQNXZ_A_004), Youth Innovation Promotion Association of Chinese Academy of Sciences (2017423), Special fund for talented persons of Sichuan provincial Party Committee Organization Department, and Key projects for foreign cooperation of International Cooperation Bureau of Chinese Academy of Sciences (182344KYSB20170009). The data were analyzed on the free online platform of Majorbio I-sanger Cloud Platform (www.i-sanger.com). ABBREVIATIONS MDG, Maotai-flavor distiller’s grains; AD, Anaerobic digestion; VFAs, Volatile fatty acids; OLR, Organic loading rate; HRT, Hydraulic retention time; TE, Trace elements; VBPR, Volumetric biogas production rate; SBPR, Specific biogas production rate; ORP, Oxidation and reduction potential; TA, total alkalinity; BA, bicarbonate alkalinity; TS, Total solid; VS, Volatile


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Anaerobic thermophilic digestion of Maotai-flavor distiller's grains

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