water

Article Impact of Organic Loading Rate on Performance and Methanogenic Microbial Communities of a Fixed-Bed ◦ Anaerobic Reactor at 4 C

Hongyan Zhao 1 , Feifan Yan 1, Xue Li 2, Renzhe Piao 1, Weidong Wang 3 and Zongjun Cui 2,* 1 College of Agronomy, Yanbian University, Yanji 133002, China; [email protected] (H.Z.); [email protected] (F.Y.); [email protected] (R.P.) 2 College of Agronomy and Biotechnology, Center of Biomass Engineering, China Agricultural University, Beijing 100193, China; [email protected] 3 School of Life Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163000, China; [email protected] * Correspondence: [email protected]; Tel.: +86-10-62731857

 Received: 26 June 2020; Accepted: 7 September 2020; Published: 16 September 2020 

Abstract: We investigated the feasibility of producing biogas in a fixed-bed anaerobic reactor at 4 ◦C with a gradual increase in organic loading rate (OLR). Reactor efficiency was highest when OLR was 4.33 kg/m3 d, whereas the reactor acidification occurred when OLR was 4.67 kg/m3 d. · · The values of methane content, biogas production, chemical oxygen demand (COD) removal rate, biogas production rate, acetic acid content, and propionic acid content were 69.3%, 5.33 L, 59.8%, 1.03 L/OLR, 0.17 g/L, and 1.15 g/L, respectively. The pH was stable and ranged from 7.2 to 6.8 when the reactor was operating at 4 ◦C during OLR increase. The 16S rRNA gene analysis revealed that the dominant were Methanosaetaceae at 30 ◦C. At 4 ◦C, the dominant archaea were , which were more abundant in adhering sludge compared to settled sludge. In conclusion, operating a fixed-bed anaerobic reactor at psychrophilic temperatures is more suitable.

Keywords: fixed-bed reactor; organic loading rate; low temperature; anaerobic reactor;

1. Introduction Anaerobic digestion, an effective treatment for wastewater containing biodegradable organic matter, reduces pollution and produces biogas for use as fuel [1,2]. Many countries have reported using anaerobic digestion for wastewater treatment, including China, the USA, Germany, Austria, Tunisia, Denmark, Italy, Spain, South Africa, and the Netherlands [3–7]. The USA and Germany are capable of producing 100% of their electricity, whereas Austria is running at an 8% surplus [4]. However, the capacity for wastewater treatment in China lags behind other countries, especially when it comes to energy recovery [8]. Despite this shortfall, clean energy production via anaerobic digestion of treated wastewater has significant potential in China. Temperature greatly influences the performance, energy efficiency, and stability of the anaerobic digestion process, especially in cold environments. Most biogas is produced under mesophilic or thermophilic conditions [9–12]. Presently, mesophilic anaerobic digestion has a higher performance and methane content, but lower pathogen sterilization efficiency. Thermophilic anaerobic digestion has a higher organic loading rate (OLR) and an increased ability to kill pathogens; however, the methane content is lower even though energy consumption is higher. The energy input required for anaerobic digestion at psychrophilic temperatures is markedly reduced due to the decreased energy needed for heating the bioreactor; thus, the operating costs for anaerobic digestion at psychrophilic temperatures are considerably reduced [2,13].

Water 2020, 12, 2586; doi:10.3390/w12092586 www.mdpi.com/journal/water Water 2020, 12, 2586 2 of 10

The viability of high-rate anaerobic wastewater treatment for reduced temperature wastewater depends on: (1) the attributes and development of seed material subjected to sub-mesophilic conditions; (2) the exceptionally high sludge retention during elevated hydraulic loading conditions, which allows very little viable biomass to escape from the reactor; (3) good interaction between retained sludge and wastewater to fully utilize the bioreactor capacity; (4) the type of organic pollutants in the wastewater; and (5) the reactor configuration [14]. The feasibility of anaerobic digestion of various wastes with psychrophilic or low-temperature (<20 ◦C) reactors has been demonstrated in a laboratory setting [15,16]. In a study focused on anaerobic degradation of winery wastewater between 4 and 10 ◦C with an upflow anaerobic sludge blanket (UASB) type of reactor, effluent chemical oxygen demand (COD) was approximately 0.1 g/L[17]. McKeown and colleagues reported that an expanded granular sludge bed-anaerobic filter (EGSB-AF) type of reactor performed stably and efficiently from 4 to 10 ◦C, with a COD removal rate of 82%, a 1 methane biogas concentration of >70%, and methane yield >4 L.d− [18]. Molasses is a typical feedstock for fermentation; however, the effluent is hard to treat. As molasses has been a common material for producing ethanol, yeast, amino acids, enzymes, etc., it is necessary to treat molasses-processed wastewater, which has a high COD concentration (50,000–100,000 mg COD/L) and low pH value (4–5) in a timely and effective manner. Additionally, significant emission of wastewater may cause catastrophic harm to the environment. Molasses wastewater contains high levels of organic compounds, cations, and anions, causing operational problems for anaerobic biological treatment. To establish a high organic loading treatment system for industrial molasses wastewater [19,20], we reported the operating parameters and methanogenic community of a fixed-bed reactor at a low temperature of 5–18 ◦C in a previous study [21]; however, previous studies have not explored the temperature threshold and the ability of fixed-bed reactors to resist OLR under the temperature threshold. Therefore, the effect of OLR on performance and methane production at 4 ◦C for anaerobic digesters has not yet been investigated. Increasing the OLR of the reactor is a necessary step to reduce the anaerobic reactor volume and the cost of engineering [2]. The purpose of this investigation was to determine the effects of increasing substrate OLR on the performance of a fixed-bed anaerobic reactor operated at 4 ◦C and establish the maximum OLR shock impact on the reactor performance. Molasses wastewater was treated using a fixed-bed reactor packed with active carbon fiber for biofilm formation. Performance parameters included pH, biogas production, volatile fatty acid (VFA) and methane content, COD removal rate, and the dynamic composition of the methanogenic community.

2. Methods

2.1. Bioreactor A laboratory-scale, fixed-bed anaerobic reactor (effective volume, 10 L), of the same design as the one described by Zhao [22] was used. The biofilm reactors contained six cylindrical carbon fiber textiles (inner diameter, 5.5 cm; height, 27 cm; thickness, 2 mm) joined with stainless steel wire. The bioreactor was positioned in an incubator (Sanyo model MIR 254, Japan) to regulate temperature [21]. Molasses wastewater (MWW) was added to the reactor with a peristaltic pump. Biogas was sampled through a porthole at the top of the reactor, and the biogas volume was determined by water displacement. Effluent (30 to 50 mL) was periodically sampled from the reactor. Bioreactor effluent and biogas were collected for the determination of chemical oxygen demand (COD) concentration, VFA content, pH, and methane (CH4) content. The COD concentration was analyzed using a Lovibond water quality monitor (model 99731COD, Germany); VFA concentration was assessed using a high-performance liquid chromatography system (Shimadzu model LC-MS2020, Japan). The Hitachi La ChromC18-AQ (5 cm) chromatographic column, column temperature 25 ◦C, mobile phase was 1 mmol/LH2SO4 and 8 mmol/L Na2SO4, mobile phase flow rate was 0.6 mL/min, sample volume was 10 µL, and the acquisition time was 60 min. The pH was determined with a pH meter (Horiba compact model B-212, WaterWater2020 2020, 12, 12, 2586, x FOR PEER REVIEW 33 of of 10 10

compact model B‐212, Japan); methane levels were quantified using a biogas analyzer (Landtec, Japan);USA).Sampling methane for levels the wereanalysis quantified of the reactor using operating a biogas parameters analyzer (Landtec, was carried USA).Sampling out every two for days. the analysis of the reactor operating parameters was carried out every two days. 2.2. Feeding Solution and Seed Sludge 2.2. Feeding Solution and Seed Sludge Artificial wastewater consisting of molasses (10 L, sag, 70%; brix, 45%), Whiskas cat food (800 g, China),Artificial and tap wastewater water (90 consisting L) had a of COD molasses concentration (10 L, sag, of 70%;100,000 brix, mg/L. 45%), The Whiskas indicated cat COD food (800concentrations g, China), andwere tap attained water by (90 diluting L) had athe COD artificial concentration wastewater of 100,000with water. mg/L. To The ensure indicated that CODmicroorganisms concentrations received were adequate attained byamounts diluting of thenitrogen artificial and wastewater phosphorus, with the ratio water. of ToCOD:N:P ensure was that microorganismskept at 300 to 500:5:1. received The adequate reactor inoculant amounts consisted of nitrogen of mesophilic and phosphorus, ally grown the ratio granular of COD:N:P sludge (4 was L) keptfrom at a 300 full to‐scale 500:5:1. Coca The‐Cola reactor production inoculant wastewater consisted treatment of mesophilic plant. ally The grown total solid granular (TS) and sludge volatile (4 L) fromsolid a (VS) full-scale contents Coca-Cola of the inoculated production sludge wastewater were 33.63% treatment and 6.16%, plant. respectively. The total solid (TS) and volatile solid (VS) contents of the inoculated sludge were 33.63% and 6.16%, respectively. 2.3. Experimental Protocol 2.3. Experimental Protocol The reactor ran at a hydraulic retention time (HRT) of six days and an initial operating temperatureThe reactor of ran30 °C. at a MWW hydraulic was retention the main time carbon (HRT) source of six for days microbial and an growth. initial operating The trial consisted temperature of oftwo 30 operation◦C. MWW stages, was thewhich main were carbon characterized source for by microbialchanges in growth. temperature The or trial OLR. consisted On day of137 two of operationthe trial, stages, the operating which were temperature characterized was bydecreased changes inby temperature 2 °C increments or OLR. every On day2 HRT, 137 of until the trial,the thetemperature operating temperaturefell to 4 °C, after was which decreased the reactor by 2 ◦C was increments stably operated every 2 HRT,for an until additional the temperature 60 days. The fell toinitial 4 ◦C, OLR after whichwas 6.67 the kg/m reactor3∙d. wasWhen stably the operating operated fortemperature an additional fell 60to 4 days. °C, the The OLR initial fell OLR to 0.67 was 3 3 6.67 kg3/m d. When the operating temperature fell to 4 C, the OLR fell to 0.67 kg/m d. To determine kg/m ∙d. To· determine the maximum OLR shock impact◦ on reactor performance (Figure· 1), the OLR theranged maximum from 0.67 OLR to shock 4.67 kg/m impact3∙d onand reactor increased performance by 0.33 kg/m (Figure3∙d with1), the every OLR 2 rangedHRT. The from pH 0.67 of the to 4.67 kg/m3 d and increased by 0.33 kg/m3 d with every 2 HRT. The pH of the MWW was maintained at MWW was· maintained at 7.0 ± 0.2 by automatic· titration with NaOH. 7.0 0.2 by automatic titration with NaOH. ±

4.67 4.33 4 3.67 3.33 /d) 3 3 2.67 2.33 OLR(kg/m 2 1.67 1.33 1 0.67

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Time(days)

FigureFigure 1. 1.Operating Operating parameters parameters of of the the fixed-bed fixed‐bed reactor reactor atat 44 ◦°C.C. OLR = organicorganic loading loading rate. rate. 2.4. Quantitative PCR of Methanogens 2.4. Quantitative PCR of Methanogens Samples of granular sludge from the fixed-bed reactor were collected at 30 C and 4 C. Adherent Samples of granular sludge from the fixed‐bed reactor were collected at 30 ◦°C and 4 °C.◦ Adherent sludge from the carbon fiber biofilm (approximately 5 mm; designated as adhering sludge) was sludge from the carbon fiber biofilm (approximately 5 mm; designated as adhering sludge) was collected directly from three active carbon fiber sheets (15 mm 30 mm 2 mm) in the reactor. Settled collected directly from three active carbon fiber sheets (15 mm× × 30 mm ×× 2 mm) in the reactor. Settled sludge was collected from valves at the bottom of the reactor. After 156 days of operation, collected sludge was collected from valves at the bottom of the reactor. After 156 days of operation, collected samples of adhering sludge and settled sludge were centrifuged (8000 g, 10 min) and the supernatants samples of adhering sludge and settled sludge were centrifuged (8000 g, 10 min) and the supernatants were removed. The resulting samples (0.3 g) were subsequently used for DNA extraction. The biomass

Water 2020, 12, 2586 4 of 10 amount required for DNA extraction was based on the volatile suspended solids (VSS) concentration. Genomic DNA for the PCR template was extracted with an automated nucleic acid extractor (BioTeke Biotech Co., Ltd., Beijing, China). Specific primer sets and 5’-nuclease probes for quantitative real-time PCR (Q-PCR) were previously described [23]. The following primers sets were used: Methanobacteriales (MBT; MBT857F, MBT929F, MBT1196R; amplicon size: 343 bp); Methanomicrobiales (MMB; MMB282F, MMB749F, MMB832R; amplicon size: 506 bp); (MSC; MSC380F, MSC492F, MSC828R; amplicon size: 408 bp); and Methanosaetaceae (MST; MST702F, MST753F, MST862R; amplicon size: 164 bp). TaqMan probes were labeled with a FAM reporter and a BHQ-1 quencher. Reactions were run on an ABI 7500 System (Model 7500, Applied Biosystems, USA). The Q-PCR reaction mix (20 µL) consisted of the following ingredients: 2 TaqMan Universal PCR Master Mix (Applied Biosystems, USA), PCR-grade × water; 10 µM primers, 1 µM TaqMan probe, and template DNA (1 µL). The PCR protocol was as follows: denaturation, 10 min at 94 ◦C; 40 cycles: 10 s at 94 ◦C, 30 s at 60 ◦C (63 ◦C for the MMB primer set). Genomic DNA was extracted from the following archaea species to produce standards for real-time PCR: Methanobacteriales (Methanobrevibacter arboriphilus NBRC101200), Methanomicrobiales ( hungatei NBRC100397), Methanosarcinaceae (Methanosarcina acetivorans NBRC100939), and Methanosaetaceae (Methanosaeta thermophila NCBR101360). Archaea samples were obtained from NITE Biological Research Center (NBRC, Chiba, Japan). PCR (primer sets described above) was used to amplify target rRNA gene sequences from each strain. After purification (TIANgel Midi Purification Kit), the PCR products were inserted into the pGEM-T Easy Vector. Standards for real-time PCR assays were generated by making 10-fold serial dilutions (102 to 109 copies per µL) of each plasmid. Standard curves were used to estimate 16S rRNA gene copy concentrations within a linear range. Granule biomass-based concentrations (copies/g granule VSS) were calculated based on copy number per µL and VSS concentration for each granular sludge sample. Duplicates for each bioreactor DNA sample were analyzed.

3. Results

3.1. Reactor Performance during the Entire Digestion Process Biogas production is an indicator of reactor performance. For example, changes in biogas production can reflect altered microbial activity and disorder caused by temperature shock [24]. Biogas production gradually increased with increasing OLR at 4 ◦C (Figure2a). When the OLR was 0.67 kg/m3 d, 1 kg/m3 d, 1.33 kg/m3 d, 1.67 kg/m3 d, 2 kg/m3 d, 2.33 kg/m3 d, 2.67 kg/m3 d, 3 kg/m3 d, · · · · · · · · 3.33 kg/m3 d, 3.67 kg/m3 d, 4 kg/m3 d, 4.33 kg/m3 d, and 4.67 kg/m3 d, the average biogas production · · · · · was 0.69 0.01 L, 0.53 0.01 L, 1.02 0.06 L, 1.16 0.06 L, 1.19 0.06 L, 1.26 0.08 L, 1.22 0.08 L, ± ± ± ± ± ± ± 1.43 0.11 L, 2.28 0.21 L, 4.12 0.15 L, 3.61 0.09 L, 4.47 0.10 L, and 2.86 0.35 L, respectively. ± ± ± ± ± ± When the OLR was between 0.67 and 3 kg/m3 d, biogas production ranged from 0.6 L to 1.6 L. · Biogas production dramatically increased to 3.7 L at an OLR of 3.33 kg/m3 d, indicating that the · reactor was operating successfully at 4 ◦C. The highest biogas efficiency was obtained when the OLR was 3.67 kg/m3 d, and the biogas production rate was 1.12 L/OLR. When the OLR was 4.33 kg/m3 d, · · the biogas production rate was 1.03 L/OLR, indicating that the fixed-bed reactor is stable (Figure2d). On day 3, biogas production increased to 6.73 L when the OLR was 4.67 kg/m3 d. This increase in · biogas production was followed by a rapid decline to 2.34 L on the fifth day of operation, indicating that the operation of the reactor has been affected when the OLR was 3; however, since the methanogenic microbial communities were suited to the microenvironment in the reactor, biogas production was increased by OLR increase and biogas production tended to gradually stabilize at an OLR of 4.33, and the reactor was soured at an OLR of 4.67. Several previous studies have reported that anaerobic fixed-bed reactors with carbon fiber biofilm have excellent performance when impacted by high organic load rate [19,21]. At 4 C, 4.33 kg/m3 d was the optimal OLR. ◦ · Water 2020, 12, x 2586 FOR PEER REVIEW 55 of of 10 10

5 7 5 80

6 70 4 4 OLR(Kg/m3/d) 60 Biogas production(L/d) 5 /d) 3 /d) 3 50 3 4 3

40 3 2 2 OLR(Kg/m OLR(Kg/m 30 Menthane content(%) 2 production(L) Biogas 1 1 OLR(Kg/m3/d) 20 1 Methane content(%) 10 0 0 0 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 0 4 8 121620242832364044485256606468727680 Time(days) Time(days) (a) (b)

1.2

5 7.5 1.0

7.0 4 ） 0.8 6.5 L/OLR （ /d) 3 3

6.0 0.6

2 pH Effluent OLR(Kg/m 5.5 OLR(Kg/m3/d) 0.4

1 Effluent pH rate production Biogas 5.0

0.2 0 4.5 0 4 8 121620242832364044485256606468727680 Time(days) 0.0 0.67 1 1.33 1.67 2 2.33 2.67 3 3.33 3.67 4 4.33 4.67 OLR(Kg/m3/d) (c) (d)

Figure 2. 2. ReactorReactor performance performance during during the the entire entire digestion digestion process. process. ( (aa)) Biogas Biogas production; production; ( (bb)) Methane Methane content;content; ( c))E Effluentffluent pH; ( d) Biogas production rate.

Figure2 b2b shows shows changes changes in the in methanethe methane content cont at dientfferent at different OLRs while OLRs the operatingwhile the temperature operating temperaturewas 4 C. At OLRswas 4 between°C. At OLRs 0.67 kgbetween/m3 d and0.67 1.33 kg/m kg3·d/m and3 d, methane1.33 kg/m content3·d, methane fluctuated content between fluctuated 35.8% ◦ · · betweenand 57.5%. 35.8% At anand OLR 57.5%. of 1.67 At kgan/ mOLR3 d, of methane 1.67 kg/m content3·d, methane increased content rapidly increased to 70.7% rapidly and the to methane 70.7% · andproduction the methane at this production time was at 0.6 this L/d. time At was OLRs 0.6 between L/d. At OLRs 3.33 kg between/m3 d and 3.33 4.33 kg/m kg3·d/m and3 d, 4.33 the methanekg/m3·d, · · thecontent methane was approximatelycontent was approximately 63.4% and the 63.4% methane and productionthe methane at production this time was at this 2.6 Ltime/d. On was operating 2.6 L/d. Onday operating 5, when OLRday 5, was when 4.67 OLR kg/ mwas3 d, 4.67 the kg/m methane3·d, the content methane was content 46.5%. was This 46.5%. indicates Thisthat indicates fixed-bed that · fixed-bedreactors with reactors carbon with fiber carbon biofilm fiber and biofilm high shock and high resistance shock produce resistance more produce methane more at low methane temperature. at low temperature.The pH is an important indicator of operating performance in the anaerobic digestion of wastewater. If theThe pH pH is toois an low important or high, theindicator normal of physiological operating performance activities of microorganismsin the anaerobic indigestion the reactor of wastewater.are inhibited If [ 25the]. pH The is optimal too low pHor high, range the for normal anaerobic physiological fermentation activities with methanogensof microorganisms is 6.0 in to the 8.0; reactorthus, the are pH inhibited of the wastewater [25]. The optimal in the anaerobicpH range reactorsfor anaerobic was maintained fermentation within with thatmethanogens specified range.is 6.0 toAt 8.0; OLRs thus, between the pH 0.67of the kg wastewater/m3 d and 4.33in the kg anaerobic/m3 d, the reactors pH was was stable maintained between within 7.2 and that 6.8 specified at 4 C, · · ◦ range.indicating At OLRs that the between fixed-bed 0.67 anaerobic kg/m3·d and reactor 4.33 was kg/m operating3·d, the pH stably was under stable this between OLR range. 7.2 and When 6.8 at the 4 °C,OLR indicating was increased that the to fixed-bed 4.67 kg/m anaerobic3 d, pH fell reactor sharply was to operating 5.5 on the stably fifth under day of this operation, OLR range. leading When to · thesouring OLR ofwas the increased reactor. Our to 4.67 results kg/m show3·d, pH that fell the sharply highest to OLR 5.5 on was the 4.33 fifth kg day/m3 ofd operation, at 4 C (Figure leading2c). to · ◦ souring of the reactor. Our results show that the highest OLR was 4.33 kg/m3·d at 4 °C (Figure 2c).

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3.2.3.2. Influent Influent and and Effluent Effluent COD COD and and COD COD Removal Removal Rate(a) Rate(a) and and VFA VFA Content(b) With with OLROLR throughoutThroughout the the OperationOperation Time Time CODCOD removal removal is is an an important important indicator indicator of of the the normal normal operation operation of of the the reactor. reactor. The The effluent effluent COD COD roserose gradually asas the the OLR OLR was was increased, increased, which which suggested suggested that that COD COD accumulated accumulated in the in digester the digester when whenthe OLR the wasOLR high was high [19,26 [19,26].]. When When the OLR the OLR was was between between 0.67 0.67 and and 1.33 1.33 kg/ kg/mm3 d,3∙ ad, gradual a gradual increase increase in · inthe the COD COD removal removal rate rate occurred occurred with with increasing increasing OLR OLR at 4at◦ 4C. °C. The The COD COD removal removal rate rate was was 69.5% 69.5% at anat anOLR OLR of 1.33of 1.33 kg/ mkg/m3 d,3 for∙d, for which which the the influent influent COD COD and and effluent effluent COD COD were were 4000 mg4000/L mg/L and 1220 and mg1220/L, · mg/L,respectively. respectively. At an OLR At an of OLR 1.67 kgof /m1.673 d, kg/m the COD3∙d, the removal COD removal rate gradually rate gradually decreased decreased to 20%, indicating to 20%, · indicatingthat the OLR that was the aOLRffected was by affected the low by temperature. the low temperature. The COD removal The COD rate removal fluctuated rate between fluctuated 35 betweenand 69.5%, 35 withand 69.5%, an increase with inan the increase OLR. Atin anthe OLR OLR. of At 4.67 an kg OLR/m3 ofd, 4.67 the COD kg/m removal3∙d, the COD rate gradually removal · ratedeclined gradually to 10% declined on the fifth to 10% day on of operation.the fifth day Our of resultsoperation. show Our that results the optimal show OLRthat the was optimal 4.33 kg /OLRm3 d 3 · wasat 4 ◦4.33C (Figure kg/m 3∙da). at 4 °C (Figure 3a).

0.7 1.6

0.6 1.4

Acetic acid 1.2 0.5 Propionate acid 1.0 0.4 0.8 0.3 0.6 Acetic acid(g/L) Acetic 0.2 Propionate acid(g/L) 0.4

0.1 0.2

0.0 0.0 0.67 1 1.33 1.67 2 2.33 2.67 3 3.33 3.67 4 4.33 4.67 OLR(Kg/m3/d) (a) (b)

FigureFigure 3. 3. InfluentInfluent and and effluent effluent chemical chemical oxygen oxygen demand demand (COD) (COD) and and COD COD removal removal rate rate ( (aa)) and and volatile volatile fattyfatty acid acid (VFA) (VFA) content content ( (b)) with OLR throughout throughout the operation time.

TheThe VFA contentcontent reflects reflects the the operation operation and and organic organic compound compound utilization utilization in the in anaerobic the anaerobic reactor reactor(Figure 3(Figureb). The 3b). acetic The acid acetic content acid increasedcontent increased from 0.15 from g /L 0.15 at 0.67 g/L kg at/ m0.673 d kg/m to 0.373∙d g to/L 0.37 at 1.33 g/L kg at/m 1.333 d. · · kg/mThe propionic3∙d. The propionic acid content acid was content 0.15 was g/L when0.15 g/L the when OLR the was OLR between was between 0.67 kg/m 0.673 d andkg/m 1.333∙d and kg/m 1.333 d. · · kg/mThe acetic3∙d. The acid acetic content acid decreased content decreased from 0.34 from g/L at 0.34 1.67 g/L kg/ atm 31.67d to kg/m 0.243 g∙d/L to at 0.24 3 kg g/L/m3 atd. 3 The kg/m acetic3∙d. The acid · · aceticand propionic acid and acid propionic contents acid fluctuated contents when fluctuated OLR was when between OLR 3.33 was kg /mbetween3 d and 43.33 kg /mkg/m3 d.3 When∙d and the 4 · · kg/mOLR3 was∙d. When 4.33 kg the/m OLR3 d, the was acetic 4.33 acidkg/m content3∙d, the wasacetic 0.17 acid g/L content and the was propionic 0.17 g/L acid and content the propionic was 1.15 acid g/L. · contentAlthough was the 1.15 pH g/L. decreased Although to 6.8, the thepH fixed-beddecreased reactor to 6.8, operatedthe fixed‐ normallybed reactor for operated 2 HRT. The normally fixed-bed for 2reactor HRT. souredThe fixed when‐bed the reactor OLR reachedsoured 4.67when kg the/m 3OLRd, since reached acetic 4.67 acid kg/m and propionic3∙d, since acidacetic production acid and · propionicexceeded acid the degradationproduction exceeded capacity the of the degradation methanogens. capacity A previousof the methanogens. report demonstrated A previous that report the demonstrateddegradation of that methanogenic the degradation propionate of methanogenic requires propionate-oxidizing propionate requires bacteria propionate and methanogenic‐oxidizing bacteriabacteria. and Compared methanogenic to other VFAs,bacteria. propionate Compared is highly to other toxic VFAs, at low propionate temperatures, is ifhighly it accumulates toxic at low [27]. temperatures,Formic acid and if it accumulates butyric acid [27]. were Formic not detected. acid and Ourbutyric results acid were show not that detected. the maximum Our results OLR show was that4.33 the kg/ mmaximum3 d at 4 C.OLR was 4.33 kg/m3∙d at 4 °C. · ◦ 3.3.3.3. Quantitative Quantitative PCR PCR of of Methanogens Methanogens QuantitativeQuantitative PCR PCR revealed revealed that that the the methanogenic methanogenic community community composition composition was was altered altered in in both both thethe carbon carbon fiber fiber carrier carrier and and settled settled sludge sludge at at 4 4 °C◦C (Figure (Figure 44).). Methanosaetaceae dominated in the seed sludgesludge at 44 ◦°C,C, and and in in the the carbon carbon fiber fiber carrier carrier and and settled settled sludge sludge at 30 ◦atC, 30 accounting °C, accounting for 95.40%, for 95.40%, 92.72%, 92.72%,and 97.26% and of97.26% the methanogens of the methanogens (sum of (sum all quantified of all quantified 16s rRNA 16s genes),rRNA genes), respectively. respectively. These resultsThese resultswere consistent were consistent with published with published reports showing reports showing a large quantity a large of quantity Methanosaetaceae of Methanosaetaceae in mesophilic in mesophilicanaerobic fermentation anaerobic fermentation of fixed-bed of reactors fixed‐bed and reactors stable granular and stable anaerobic granular digesters. anaerobic digesters.

Water 2020, 12, 2586 7 of 10 Water 2020, 12, x FOR PEER REVIEW 7 of 10

Figure 4. Quantitative PCR of methanogens in adhering sludge and settled sludge. Granular Figure 4. Quantitative PCR of methanogens in adhering sludge and settled sludge. Granular biomass biomass samples from seed inoculum, adherent sludge, and settled sludge were assayed. S = sample samples from seed inoculum, adherent sludge, and settled sludge were assayed. S = sample of seed of seed sludge; TM = adherent sludge; TD = settled sludge; TDS = sludge sample after reactor sludge; TM = adherent sludge; TD = settled sludge; TDS = sludge sample after reactor souring; MBT souring; MBT = Methanobacteriales; MSC = Methanosarcinaceae; MMB = Methanomicrobiales; = Methanobacteriales; MSC = Methanosarcinaceae; MMB = Methanomicrobiales; MST = MST = Methanosaetaceae. Methanosaetaceae.

In both the carbon fiber carrier and settled sludge at 4 ◦C, 16S rRNA gene concentrations for In both the carbon fiber carrier and settled sludge at 4 °C, 16S rRNA gene concentrations for Methanomicrobiales were significantly higher than at 30 ◦C. At 4 ◦C, the Methanomicrobiales 16S Methanomicrobiales were significantly higher than at 30 °C. At 4 °C, the Methanomicrobiales 16S rRNA gene concentrations were 99.78% and 99.91% more abundant in the carbon fiber carrier and rRNA gene concentrations were 99.78% and 99.91% more abundant in the carbon fiber carrier and settled sludge, respectively, compared to the concentrations at 30 ◦C. settled sludge, respectively, compared to the concentrations at 30 °C. Methanomicrobiales 16S rRNA gene concentration was higher in the carbon fiber carrier compared Methanomicrobiales 16S rRNA gene concentration was higher in the carbon fiber carrier to the settled sludge at 4 C (86.28% vs. 63.06% of the methanogenic population). This finding is compared to the settled ◦sludge at 4 °C (86.28% vs. 63.06% of the methanogenic population). This consistent with published reports showing that Methanomicrobiales tended to adhere to carbon fiber finding is consistent with published reports showing that Methanomicrobiales tended to adhere to carriers in a fixed-bed reactor, as well as reports demonstrating the dominance of methanogens under carbon fiber carriers in a fixed‐bed reactor, as well as reports demonstrating the dominance of psychrophilic conditions [21,28,29]. methanogens under psychrophilic conditions [21,28,29]. 4. Discussion 4. Discussion Many studies on the treatment of wastewater using various bioreactors focused on the low Many studies on the treatment of wastewater using various bioreactors focused on the low operating temperature of 4 C[17,18,20–33]. Nevertheless, few reports have discussed the effect of operating temperature of 4◦ °C [17,18,20–33]. Nevertheless, few reports have discussed the effect of different OLRs on reactor operation in a fixed-bed anaerobic reactor operated at 4 C. In this study, different OLRs on reactor operation in a fixed‐bed anaerobic reactor operated at 4 °C.◦ In this study, we treated molasses wastewater with increasing OLRs from 0.67 kg/m3 d to 4.67 kg/m3 d at 4 C. we treated molasses wastewater with increasing OLRs from 0.67 kg/m3∙d to· 4.67 kg/m3∙d at· 4 °C. The◦ The maximal treatment OLR of the fixed bed anaerobic reactor were 4.33 kg/m3 d at 4 C; the COD maximal treatment OLR of the fixed bed anaerobic reactor were 4.33 kg/m3∙d· at 4 °C;◦ the COD removal rate was approximately 60% at this OLR (Figure3a), demonstrating the improved e fficiency removal rate was approximately 60% at this OLR (Figure 3a), demonstrating the improved efficiency of biogas production in a fixed-bed anaerobic reactor compared to other reactors. The treatability of of biogas production in a fixed‐bed anaerobic reactor compared to other reactors. The treatability of winery wastewater by a UASB reactor was 1.7 g/L d at 4 C, with a removal rate of 57% [17], whereas winery wastewater by a UASB reactor was 1.7 g/L·∙d at 4 ◦°C, with a removal rate of 57% [17], whereas the treatability of a VFA mixture by an EGSB reactor was 5 g/L d at 4 C, with a removal rate >90% [33]. the treatability of a VFA mixture by an EGSB reactor was 5 ·g/L∙d at◦ 4 °C, with a removal rate >90% Additionally, the treatability of VFA wastewater by an EGSB-AF bioreactor was 3.75 kg/m3 d at 4 C, [33]. Additionally, the treatability of VFA wastewater by an EGSB‐AF bioreactor was 3.75 kg/m· 3∙d ◦at with4 °C, a with removal a removal rate > rate85% >85% [18]. [18]. Molasses Molasses wastewater wastewater contains contains many many compounds compounds and and aa high COD COD concentrationconcentration of of mineral mineral salts salts and and has has aa badbad smellsmell andand dark brown color, and is therefore of of serious serious environmentalenvironmental concern concern and and is is hard hard to to treat. treat. There There is is aa highhigh potential for the further utilization utilization of of such such wastewater.wastewater. However, However, to to establish establish a a high high organic organic loadingloading treatmenttreatment system for industrial molasses wastewater,wastewater, which which it it produce produce biogas biogas often often most most requiresrequires need mesophilic or or thermophilic thermophilic conditions, conditions, andand COD COD removal removal rate rate is is approximately approximately 60–80%60–80% [[19,34],19,34], andand wewe previously reported that that the the COD COD removalremoval rate rate is is approximately approximately 50% 50% at at 18 18◦ °CC[ [21].21]. InIn summary,summary, thethe fixed-bedfixed‐bed reactor was suitable suitable for for

Water 2020, 12, 2586 8 of 10 treating wastewater with a high COD concentration at low temperature and has more advantages compared with other types of reactors. The major VFAs formed during anaerobic biodegradation of wastewater rare acetic acid, propionic acid, and butyric acid. Propionate accumulation in an anaerobic reactor indicates an unstable anaerobic process; bacteria that degrade propionate are more temperature sensitive than bacteria that degrade acetate, thereby leading to propionate accumulation [35]. Additionally, the accumulation of propionate is a major contributor to reactor souring at psychrophilic temperatures [18]. At an OLR of 4.67 kg/m3 d, · propionic acid (1.48 g/L) accumulated, while pH fell to 5.5, and the COD removal rate was approximately 10%, demonstrating that the fixed-bed anaerobic reactor was soured at this OLR. In this study, the pH decreased when the OLR was 1.67 kg/m3 d. At an OLR of 2 kg/m3 d, acetate and · · propionate contents were 0.346 g/L and 0.614 g/L, respectively. Biogas production and methane content also increased. When the OLR was 3.33 kg/m3 d, the acetate content was 0.328g/L, pH decreased, biogas · production increased, and the methane content was 70.7%, this phenomenon shows that the operation of the fixed-bed reactor is impacted under this OLR, and the VFA was accumulation. The significant accumulation of VFAs in the reactor (>150 mg/L) was the first warning sign that the digester was no longer performing at peak efficiency. Acetate and propionate degradation are susceptible to low temperatures [33]. Due to the low-temperature inhibition of acetogenic bacteria, which resulted in high acetate concentration (300 mg/L), VFA accumulation ultimately led to bioreactor failure, or “souring” [35,36]. Therefore, the highest biogas production efficiency was at an OLR 3.67 kg/m3 d, which further confirms that the stable · operation of the reactor was at an OLR of 3.33 kg/m3 d, and the fixed-bed reactor has a strong shock · resistance when operating at 4 ◦C. Previous studies have shown that Methanomicrobiales undergo hydrogenotrophic methanogenesis and are critical for low-temperature anaerobic digestion. Methanomicrobiales adhere easily to carbon fiber and fixed-bed reactors use carbon fiber as biofilm carriers [21]. Thus, microbial activity is increased and microbial washout is prevented under psychrophilic conditions. In this study, the concentration of Methanomicrobiales 16S rRNA genes in the fiber carbon and settled sludge increased by approximately 52.23-fold and 185.23-fold at 4 ◦C compared to 30 ◦C. Therefore, Methanomicrobiales are the dominant methanogens at the low temperature of 4 ◦C. Previously published reports show that retention of hydrogen is elevated at psychrophilic temperatures because of increased gas solubility. If methanogens are at low temperatures, hydrogen is metabolically and thermodynamically more advantageous than acetate. Therefore, at low temperatures, fixed-bed reactors have better methane production (the methane content was approximately 70%) (Figure2b).

5. Conclusions In this study, the fixed-bed anaerobic reactor was high shock resistance for treating high-concentration organic wastewater at 4 ◦C. The largest wastewater treatment capacity occurred at 4.33 kg/m3 d OLR. Biogas production peaked when the volume and COD removal rates were 3.7 L · and 69.5%, respectively, at OLRs of 3.33 kg/m3 d mg/L and 1.33 kg/m3 d, respectively. The highest · · methane content was 70.7%, and the pH remained stable between 6.8 and 7.2. Methanomicrobiales, which preferentially adhere to carbon fiber carriers, were the dominant methanogens at 4 ◦C, and play an important role in anaerobic granular sludge systems.

Author Contributions: Conceptualization, H.Z. and Z.C.; Methodology, X.L. and F.Y.; Software, R.P., X.L. and W.W.; Validation, H.Z., F.Y., X.L., R.P. and Z.C.; Formal Analysis, X.L., F.Y.; Writing-Original Draft Preparation, R.P., X.L. and W.W.; Writing-Review and Editing H.Z. and Z.C.; Supervision, Z.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Science Foundation of China (No. 51741809), Special Fund for Agro-Scientific Research in the Public Interest (No. 201503137), the National Key Technology Research and Development Program of China (No. 2015BAD21B04), Key projects of Natural Science Foundation of Heilongjiang Province (ZD2018005), and Open Foundation of the Heilongjiang Provincial Key Laboratory of Environmental Microbiology and Recycling of Argo-Waste in Cold Region of China (No. 201711). Conflicts of Interest: The authors declare no conflict of interest. Water 2020, 12, 2586 9 of 10

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