Effect of effluent recirculation rate on the performance of anaerobic bio-filter treating coal gasification wastewater under co-digestion conditions

Abstract

In this paper, anaerobic biofilters (AF) were adopted for anaerobic co-digestion of potato starch wastewater (PSW) and coal gasification wastewater (CGW). A modified Gompertz equation was used for the analysis. With a ratio of PSW : CGW at 1 : 1 for the influent, it remarkably improved the removal effect of (chemical oxygen demand) COD and total phenol. With an influent concentration of COD of 2600 mg L⁻¹ and total phenol 200 mg L⁻¹, the effluent COD and total phenol could be decreased to 1100 mg L⁻¹ and 100 mg L⁻¹, respectively. The methane production rate was generally above 260 (mL CH₄ per g per d). In order to further enhance the performance of the system, the strategy of effluent recirculation on co-digestion was adopted. The result indicated that under a low effluent recirculation rate of 0.5, concentrations of COD and total phenol in effluent could reach as low as 800 and 70 mg L⁻¹, respectively. Also, the methane production rate was around 300 (mL CH₄ per g per d). High-throughput sequencing showed the microbial phyla Bacteroidetes, Chloroflexi, Euryarchaeota, Firmicutes dominanted the system and the abundance variation was observed at different effluent recirculation rates.

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1. Introduction

Nowadays, coal gasification technology has been rapidly developed in China due to natural gas shortage.¹ However, this technology produces a large amount of coal gasification wastewater (CGW) which contains many complex, toxic and refractory pollutants such as phenolics, heterocyclics, ammonia and sulfide.^2–4 Although, phenols and ammonia can be removed by solvent extraction and steam stripping pretreatment, some high concentration pollutants still remain in the effluent.^5–8 Due to its low biodegradability the treatment of CGW is a difficult task.

Anaerobic biological technologies are characterized by cost-effective and high efficiency in disposing refractory organic substances and could enhance the biodegradability of wastewater.^9–12 The carcinogenic hydroquinone in CGW was degraded by using anaerobic shaker test, the test indicated that the system could degrade COD and total phenol effectively and remain in a stable level when the operational parameters were changed.¹³ Advanced anaerobic expanded granular sludge bed (AnaEG) was developed for the anaerobic treatment of CGW, it needs less accessory equipment and lower power consumption, and improves the treatment efficiency.¹⁴ With a two-stage anaerobic system as a control, the two-phase anaerobic digestion of real CGW was discussed. The results indicated that the two-phase anaerobic digestion improved both anaerobic and aerobic biodegradation of CGW.¹⁵

However, it remains difficult to realize resourcezation of CGW for conventional biologic technology, such as methanogens are very vulnerable and difficult to be enriched in the system. Moreover, the toxic pollutants in CGW could restrain the activity of the functional bacteria. In order to solve these problems, AF (anaerobic biofilters) was developed to reduce the inhibition of toxic and refractory compounds to anaerobic microorganism activity and increase the biodegradability of CGW. The filters filled in the reactor not only provided additional surface area for bacterial attachment, enrichment and growth, but also promote the stability and tolerance to shock loading.¹⁶ Moreover, the spatial distribution of microflora in AF benefits in the degradation of toxic pollutants. For example, Yang¹⁷ et al. utilized a new AF reactor to treat terephthalic acid wastewater with the effluent COD concentration of less than 60 mg L⁻¹. However, there seemed to exist a maximum tolerance of anaerobic microorganism in AF to toxic substances in CGW. It is a bottleneck of CGW anaerobic treatment to alleviate the toxicity of CGW in order to improve CGW treatment efficiency in AF.

Recently, anaerobic co-digestion has been applied to treat refractory or toxic substances, as it is difficult for anaerobic bacteria to directly utilize the refractory substances as carbon source or energy, while other co-substrate easily utilized carbon source or energy exist in influent, the refractory pollutants could be degraded efficiently in the system. During co-digestion of intermediately-aged landfill leachate with sewage sludge at a ratio of 1 : 20, both biogas and methane production increased by 13% and 16.9%, respectively, in comparison with sludge treatment alone.¹⁸ A treatment process that combines the co-digesting cheese whey with manure and under a constant hydraulic retention time of 2.2 days, when increasing proportion of cheese whey in the feed, the system indicated a stable operation up to a 75% cheese whey in the feed, with an organic loading rate of 19.4 kg COD per m³ per d. Obtaining 94.7% COD removal rate and methane production rate of 6.4 m³ CH₄ per m³ per d.¹⁹ Also, anaerobic co-digestion was investigated by methanol addition to improve the biodegradation for CGW.¹¹ The corresponding maximum COD and phenol removal rates were 71% and 75%, respectively, with methanol concentration of 500 mg COD per L for a total organic loading rate of 3.5 kg COD per m³ per d and a phenol loading rate of 0.6 kg per m³ per d. Hence, finding a suitable and economic co-substrate is a good solution for toxic and refractory pollutants degradation in CGW.

Potato industry has developed rapidly in recent years and formed the industrialization base in Ningxia, Gansu, Mongolia and other provinces in China, which result in discharging potato starch wastewater (PSW) more than 8 million ton a year. Therefore PSW, as an easily biodegradable substrate, can be used as an inexpensive and economical product in the coal gasification industry. Hence, it is feasible to enhance the anaerobic biodegradability of CGW with the addition of PSW.

There are several advantages of anaerobic co-digestion as it can improve the biodegradability of the wastewater, increase the treatment efficiency of the system, optimizes the performance of AF, especially enhancing the removal efficiency of the toxic recalcitrant pollutants and finally it improves methane production. The strategy of effluent recirculation on the basis of co-degradation in AF was adopted. It was reported that effluent recirculation could reduce toxic substrate concentration in influent and speed up the upflow velocity (V_up), promote the biological exposure, improve AGS (anaerobic granular slugde) adsorption ability.

Up to date, few researches have been reported for the effluent recirculation on anaerobic co-digestion in AF treating CGW. The aim of this study was to explore the optimum ratio of CGW and PSW in influent by using a modified Gompertz equation, also the treatment efficiency of AF with and without PSW addition in influent was investigated. Furthermore, the effluent recirculation on co-digestion was adopted to explore the effect of total phenol degradation and methane production. In addition, the influence of different effluent recirculation rates on the particle size distribution (PSD) of anaerobic granular sludge (AGS), substrate utilization rate (SUR) and specific methanogenic activity (SMA) were analyzed and high-throughput sequencing was applied to analyze and investigate the dominant microorganisms during the effluent recirculation step.

2. Methods and materials

2.1 Anaerobic biofilter reactor

The AFs were made of cylindrical plexiglass and filled with soft filler. The influent was pumped into the bottom of the reactor and flowed out from the top. One of the AF (called the supplemented AF) was operated by adding PSW in the influent as co-substrate. Meanwhile, the other AF (called the control AF) was operated without adding PSW. The effective volume of the supplemented reactor and the control reactor were 2 L and 3.9 L, respectively. Both the two reactors were operated at temperatures of 35 °C and kept at same HRT of 48 h, the pH was controlled at 6.8–7.2. The produced biogas in the reactors was pretreated by 3 M NaOH absorption and then collected by a gas collection tank. The volume of CH₄ was monitored through gas flow meter. The effluent of the supplemented reactor overflowed from reactor and was collected in a recirculation pool. A recirculation pump was used to recycle a certain number of effluent according to the demands of experiment.

2.2 Inoculum

The inoculated anaerobic activated sludge was taken from an expanded granular sludge bed (EGSB) treating starch wastewater, inoculation volume was about 30% of the effective volume of each AF. The granular sludge was black with good settlement characteristics. The suspended solids (SS) and volatile suspended solids (VSS) in the reactors were about 8.57 and 5.36 g L⁻¹, respectively.

2.3 Coal gasification wastewater

CGW was taken from Harbin Coal Chemical Industry Co, Ltd in China, and it was pretreated by phenol extraction and ammonia stripping. The composition of raw CGW in this study is as follows: COD (1500–2800 mg L⁻¹), biochemical oxygen demand: BOD₅ (10–23 mg L⁻¹), total phenol (200–420 mg L⁻¹), NH₄⁺–N (180–240 mg L⁻¹), pH (6.8–8.2), total carbon: TC (720–860 mg L⁻¹), inorganic carbon: IC (90–130 mg L⁻¹), total organic carbon: TOC (650–745 mg L⁻¹).

2.4 Potato starch wastewater

The PSW was artificially prepared by grinding potatoes using grinding machine meanwhile a little sulfate was also added. The concentration of PSW was depended on the demands of experiment.

2.5 Substrate utilization rate and specific methanogenic activity test

Substrate utilization rate (SUR) of phenol and specific methanogenic activity (SMA) tests were carried out in 250 mL sealed bottles with a working volume of 200 mL. The seed sludge was fetched from the supplemented reactor. The substrates of phenol (in SUR test) and PSW (in SMA test) were controlled at the concentrations of 400 mg per L and 2400 mg COD per L, respectively. The pH in the system was controlled to 6.8–7.2 by NaHCO₃. The values of SUR and SMA were expressed as (mg phenol per g VSS per d) and (mg COD-CH₄ per g per VSS per d) respectively. N₂ (99.9%) was flushed into the bottles for 30 min and were performed at 35 ± 1 °C in swing bed with 120 rpm. All batch tests had been repeated for three times.

2.6 Analytical methods

COD, BOD₅, NH₄⁺–N, pH and MLSS were measured according to the standard procedures.²⁰ TOC, IC and TC were monitored by the total organic carbon analyzer (TOC-LCSH, Shimadzu, Japan). The volume of biogas production was determined by wet glass flowmeter, and methane content was analyzed through a 3 M NaOH solution. The concentration of total phenol was measured by the titration method.²¹ The measurements of AGS were using a laser particle size analyzer (Mastersizer 3000, UK). The scanning electron microscopy (SEM) (Digital SEM Leica 440 at 20 kV, China) was used to observe the form of AGS after sample pretreatment.

2.7 Kinetic measurements and data analysis

Batch tests with the inoculation sludge were implemented to investigate the relationship between CH₄ production and time. Firstly, four sealed bottles, namely B1, B2, B3 and B4 were prepared and added with a small amount of anaerobic activated sludge, respectively. The PSW was prepared with potato starch and controlled concentration of 2 g COD per L and pH of 6.8–7.2. In addition, 100 mg L⁻¹ of NH₄Cl was also added in it. Effective volume of the mixture in every bottle was controlled at 200 mL. The PSW and CGW were mixed with the ratios of 1 : 0, 3 : 1, 1 : 1 and 1 : 3. Then put the mixture into B1, B2, B3 and B4, respectively. N₂ (99.9%) was flushed into the bottles for 30 min. The batch tests were carried out at 35 ± 1 °C in swing bed with 120 rpm. The methane production was recorded after every 2 hour. Every experiment had been repeated for three times.

The cumulative biogas production (P) was fitted to a modified Gompertz equation²² which is a proper model for describing the progress of cumulative methane production in a batch experiment.

P: cumulative methane yield (mL); P_max: maximum methane yield (mL); R_m: maximum methane production rate (mL h⁻¹); t: digestion time (h); e: mathematical constant 2.718282; λ: lag phase (h).

2.8 High-throughput sequencing

High-throughput sequencing was used to analysis the microbial communities in AGS, checking how the microbial community changes under the different effluent recirculation rates when the performance of the supplemented reactor was in stability. The activated sludge samples at each effluent circulation rates were collected and stored at −80 °C before use. DNA was extracted as described previously with minor modification.^23,24

The extracted DNA was amplified by using a set of primers with 341F primer (CCTACACGACGCTCTTCCGATCTNCCTACGGGNGGCWGCAG) and 805R primer (GACTGGAGTTCCTTGGCACCCGAGAATTCCAGACTACHVGGGTAT CTAATCC) targeting the V3–V4 region of microbial 16S rRNA gene. The PCR conditions were set as follows: initial denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, 2.5 μL first-step PCR production is as template of second-step PCR, initial denaturation at 95 °C for 3 min, and then 5 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s and then final extension at 72 °C for 5 min. The PCR products were determined by next generation sequencing using Miseq Illumina in SKLMM Laboratory in Shanghai Jiao Tong University (Shanghai, China). To obtain the effective sequencing data, raw pyrosequencing results were processed by the following method: all high-quality sequences were extracted, aligned in greengenes to remove sequences with less than 75% identity with bacteria. OTU classification was performed using UPARSE,²⁵ briefly, high-quality sequences were dereplicated into unique sequences and then sorted by decreasing abundance, and singletons were discarded, representative non-chimeric OTU sequences were next picked. Further reference-based chimera detection was performed using UCHIME²⁶ against the RDP classifier training database. The OTU table was finalized by mapping quality-filtered reads to the remaining OTUs with the Usearch global alignment algorithm at a 97% cutoff. The most abundant sequence of each cluster was subjected to online RDP classifier (Version 2.10) for taxonomical assignment with a bootstrap cutoff of 50% (http://rdp.cme.msu.edu/classifier/classifier.jsp). The representative sequences, together with the abundance data (normalized for each sample), were used for further analysis. The alpha and beta diversities were performed using QIIME (version 1.8).²⁷

3. Results and discussion

3.1 Kinetic study of optimal volume ratio of PSW and CGW

A modified Gompertz equation was introduced in order to find out the maximum methane production in shortest time under co-digestion condition then to explore the optimal ratio of PSW and CGW in influent for the following lab-scale experiment. As shown in the Fig. 1, non-linear fitting was performed on the experimental data by using the Origin 8.0 software. The Table 1 demonstrated that the fitting constants r² were all above 0.96, indicating good correlations. The estimated parameters derived from the fitted model were exhibited. The increasing concentrations of NH₄⁺–N and total phenol in the mixture to a certain degree can stimulate the growth of methanogens and conduct to produce methane rapidly.²⁸

Fig. 1 Nonlinear regression fit of different ratio of PSW and CGW obtained from batch tests. (a) Fitting curving of ratio of PSW and CGW was 1 : 0; (b) fitting curving of ratio of PSW and CGW was 1 : 1; (c) fitting curving of ratio of PSW and CGW was 1 : 3; (d) fitting curving of ratio of PSW and CGW was 3 : 1. Error bars were obtained based on three replicates.

Table 1 The concentrations of COD, NH₄⁺–N, total phenol and pH in different volume ratio of PSW and CGW and kinetic parameters fitting by the modified Gompertz model

Ratio	1 : 0	3 : 1	1 : 1	1 : 3
Pmax: maximum methane yield (mL); Rm: maximum methane production rate; λ: lag phase; r^2: coefficient of determination; ND, not detected.
COD (mg L^−1)	2000	2215	2366	2437
Total phenol (mg L^−1)	ND	43	196	215
NH4^+–N (mg L^−1)	100	153	283	302
pH	7	7.1	7.2	7.5
Pmax (mL)	141.26	66.87	28.84	17.69
Rm (mL h^−1)	12.15	3.95	1.72	0.97
λ (h)	6.77	3.54	0.78	0.06
r^2	0.996	0.969	0.986	0.975

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Therefore, when ratios of PSW and CGW reached 1 : 1 and 1 : 3, λ were 0.78 h and 0.06 h, respectively. Which were shorten than the values (λ) under the ratios of PSW and CGW to 1 : 0 and 3 : 1. While maximum methane production rate (R_m) under the ratio of 1 : 1 was 1.72 mL h⁻¹ which was more than the value (R_m) of the ratio of 1 : 3. Hence, the optimal volume ratio of PSW : CGW was 1 : 1 in influent and adopted in the next lab scale study.

3.2 Performance of AF in disposing CGW on co-digestion

As shown in Fig. 2, the control AF was operated continuously with a constant HRT of 48 h and pH of 6.8–7.2. The variation concentrations of influent COD and total phenol resulted in the fluctuation of the effluent COD and total phenol. The average influent COD and effluent COD were about 2343 and 1599 mg L⁻¹, respectively. The removal rate of COD was about 30% and the average influent total phenol and effluent total phenol were about 300 and 200 mg L⁻¹, respectively. With the removal rate of total phenol was about 34%.

Fig. 2 Performance of the control AF, (a) removal of total phenol in control AF; (b) removal of COD in control AF.

In the supplement AF, the PSW (control the COD = 2000 mg L⁻¹) added into CGW and kept the volume ratio of PSW and CGW was 1 : 1 in influent. As shown in Fig. 3a and b, the effluent COD and total phenol remained in a stable level at HRT of 48 h and pH of 6.8–7.2, especially providing with the average removal efficiency of COD above 58%, which indicated the good stability of the system. Moreover, the effluent total phenol decreased to 100 mg L⁻¹, the result showed on the one hand, the adding PSW diluted influent concentration of the toxic pollutants in CGW and decreased the phenol loading rate in influent. Thus it can reduce restrain of toxic compounds in anaerobic digestion process²⁹ and improve balance of nutrients^30,31 and increase gas yields.³² On the other hand, low concentration of toxic pollutants can be conducive to the growth of anaerobic micro-organism especially methanogens. Hence, the methane production rate was greatly improved in supplemented reactor. As shown in Fig. 3c, after 3 days the methane production rate was rapidly increased to 260 (mL CH₄ per g per d), and then fluctuated for a while generally around 250 (mL CH₄ per g per d).

Fig. 3 Performance of the supplemented AF, (a) COD removal of the supplemented AF; (b) total phenols of the supplemented AF; (c) methane production of the supplemented AF.

The results indicated that the appropriate adding of PSW as co-substrate can induce the required enzymes of metabolism and generate enough energy to drive the initial transformation of the toxic matters²⁸ and refractory pollutants such as phenolics, heterocyclics, ammonia and sulphide and so on, which lead to poor biodegradability of CGW. When co-substrate exists in the system, it can dilute the concentrations of these toxic and refractory compounds, improve balance of nutrients and develop the synergistic microbial consortia.³³ Moreover, these toxic and refractory compounds can be transformed by co-digestion then be utilized by anaerobic micro-organism, it can be helpful in establishing the metabolic equilibrium for the enhancement of methanogenic bacteria activity²⁸ and the improvement of the anaerobic biodegradation of CGW. Hence, the methanogens can grow and reproduce rapidly. However, when the HRT was increased from 48 h to 64 h, the removal rates of COD and total phenol was not improved obviously and methane production rate was not enhanced. Although extending HRT from 48 h to 64 h, the organic loading rate and phenol loading rate were decreased to some extent. The concentrations of toxic pollutants in the system still restrained the reproduction of anaerobic micro-organism. In order to improve the treatment efficiency of the system, maintaining a certain concentration and activity of anaerobic micro-organism is indispensable. Secondly, some non-biodegradable organic matter may be existed in the CGW. Hence, only prolonging the HRT, the toxic pollutants could not biodegrade completely and the toxicity is not so easily to conquer, the performance of COD and total phenol removal and methane production rate were not enhanced obviously.

Although the toxic pollutants in CGW were diluted and degraded to some extent in co-digestion condition, the concentrations of residual toxic pollutants still exceeding an upper limit of the tolerance of anaerobic microorganisms in AF. Therefore, it is feasible to obtain better performance by further reducing restrain and improving the metabolism of methanogens in system. Hence, further investigation related to effluent recirculation in supplemented AF under co-digestion condition was carried out.

3.3 Enhanced performance of effluent recirculation on co-digestion

3.3.1 Pollutants removal. The effluent recirculation rates (R) of 0 : 1, 0.5 : 1, 1 : 1, 1.5 : 1 were discussed in supplemented AF and still kept co-digestion ratio of CGW and PSW was 1 : 1 in influent and pH in the system was controlled about 6.8–7.2. As shown in Fig. 4, comparing with the effluent recirculation rate (R) of 0 : 1, the performance of COD removal and methane production rate were remarkably enhanced when R reached 0.5 : 1, the effluent COD reached 800 mg L⁻¹ and methane production rate was increased to about 300 (mL CH₄ per g per d). Furthermore, the removal rate of total phenol was increasing obviously. Even the effluent total phenol was gradually decreasing to 70 mg L⁻¹. However, further increasing recirculation rate, the removal effects of COD and total phenol were reduced and even the methane production rate could not detected at R = 1.5. Result indicated that low effluent recirculation rate on co-digestion firstly could further reduce the concentrations of toxic pollutants, improve biodegradability in influent, enhance the adsorption ability of sludge. Moreover, the addition of the PSW as co-substrate in the recirculation operation may benefit for the growth of anaerobic micro-organism, especially for some functional bacteria such as phenol-degrading bacteria. Secondly the fillers in the reactor was beneficial to the growth and biomass accumulation in the system, while superabundant biomass would not be beneficial for degradation of pollutants.³⁴ Hence, an appropriate recirculation can scour off and remove the aged and decayed biomass of biofilms and maintain the activity of anaerobic microorganisms in the system³⁶ and the biodegradability of the effluent was greatly improved, then it will be further disposed by aerobic treatment such as AMBR (aeration membrane bioreactor).

Fig. 4 Performance of the supplemented AF with the increasing effluent recirculation rate (R), (a) removal of COD in supplemented AF with different R; (b) removal of total phenol in supplemented AF with different R; (c) methane production rate in supplemented AF with different R.

However, excessive recirculation rate could lead to high speed upwelling which could destroy the structure of AGS and the gradient distributions of anaerobic bacterial was seriously affected.³⁵ Moreover, maintaining a certain concentration of biomass in the anaerobic system was also a key to decide pollutants removal efficiency.²⁸ Nevertheless a large number of biomass was washed out from the system because of the excessive recirculation rate which leaded to unsatisfied performance.

3.3.2 Anaerobic granular sludge. Due to the adopted strategy of effluent recirculation, low recirculation rate can improve the collision and friction strength among anaerobic granular sludge (AGS), which promoted the compactness of AGS.²⁸ On the contrary excessive recirculation could destroy the AGS stability, as shown in Fig. 5a. It was indicated that when the recirculation rates reached 0 : 1 and 0.5 : 1, the PSD of 400–800 μm and 800–1200 μm dominated the main part, but when the effluent recirculation rate was increased, the PSD percent of 0–400 μm was increased gradually. The Fig. 5b demonstrated the SEM images of the AGS which fetched from the supplemented AF under different effluent recirculation rates. Before inoculation the seed appeared irregular spheres or ellipsoids with a smooth surface. However after long time cultivation by the CGW, the surface of AGS become rough and some fragments adhere to it. Because the toxic pollutants in the system could destroy the dynamic equilibrium of microorganism even contribute to a few disintegration of AGS. Secondly, slight alkaline in CGW has a certain influence on the stability of AGS.²⁸ In addition, the adversity effect of excessive effluent recirculation also contributed to this phenomenon. Moreover, the degree of fracture in AGS becomes more and more prominent with the increasing effluent recirculation rates.

Fig. 5 (a) Particle size distribution (PSD) of AGS under different effluent recirculation ratios and (b) SEM images of anaerobic granular sludge samples.

3.3.3 Substrate utilization rate and SMA tests. The results of substrate utilization rate (SUR) and specific methanogenic activity (SMA) tests were presented in Table 2. The SUR was 180 ± 5 mg phenol per g per VSS per d in R = 0.5 while when R = 1, it decreased sharply. The SMA has been observed and displayed the highest value in R = 0.5 in the system. The result showed that low recirculation rate can stimulate growth of hydrolytic acidification bacteria and methanogens. Also, the addition of PSW as co-substrate in influent facilitated degradation of toxic and refractory compounds. All of these benefited for the reproduction of the microorganisms. Hence, low recirculation rate under co-digestion could further enhance not only the phenol utilization but also the specific methanogenic activity of the anaerobic micro-organism in the system. In the lab-scale experiment when R = 1.5, there was nearly no methane production. As the excessive effluent recirculation changed the anaerobic microorganism distribution,³⁶ interfering with the co-metabolic reactions in microbial community.²⁸ Moreover, some biomass was washed out from the system because of V_up. Hence, it is difficult to keep the high concentration of methanogens. While the purpose of the SMA test is only to detect the activity of the methanogens in anaerobic sludge and the substrate of the test was only PSW. Furthermore, there was no interference of upflow velocity in the test. The inhibition of toxic pollutants was temporary and reversible.³⁶ Thus, the activity of methanogens could be recovered to some extent and a small amount of methane could be produced.

Table 2 Results of substrate utilization rate (SUR) and special methanogenic activity (SMA) tests^a

R	SUR mg phenol per g per VSS per d	SMA mg COD-CH4 per g per VSS per d
a R: effluent recirculation rate.b Values represent the mean values ± the standard error from the triplicate test samples.
0	149 ± 10^b	76 ± 12
0.5	180 ± 5	188 ± 8
1	79 ± 7	152 ± 11
1.5	46 ± 12	67 ± 12

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3.3.4 Bacterial community and diversity at different effluent recirculation rates. There are two opposite effects on effluent recirculation on anaerobic microorganism in AF. On the one hand, effluent recirculation could reduce the concentrations of toxic pollutants in the system and facilitate the proliferation of anaerobic microorganism. On the other hand, effluent recirculation could effect the gradient distribution.³⁶ The microbial community of the sludge from recirculation rates of 0, 0.5, 1.0, 1.5 were analyzed by high-throughput sequencing technology. Analytical results indicated that the predominant microbial phyla were Bacteroidetes, Chloroflexi, Euryarchaeota, Firmicutes.

The bacteria from Chloroflexi as shown in Fig. 6, was as high as 27.1% in abundance in the seed but decreasing to 19.5% in R = 0 due to the inhibition of toxic pollutants in the system, while increasing the recirculation rate, it improved gradually, even reached the value of 37.1% when the ratio was 1.0. It is indicated that the bacteria from Chloroflexi was enriched due to dilution and stimulation effect of effluent recirculation and the same conclusion applied to the microorganism from Euryarchaeota, which include many species of methanogen. The result indicated that effluent recirculation can benefit the growth of the methanogens, even the abundance was increased with the increasing effluent recirculation rates.

Fig. 6 (a) and (b) Abundance of the major bacterial groups in samples with different effluent recirculation rate (R).

Among the genera, Longilinea suggest that they likely played an important role in the methanogens associated with phenol degradation.³⁷ Such as Longilinea arvoryzae which grows between 30 °C and 40 °C, was observed at pH 5.0–8.5, with optimum growth at pH 7.0. Growth is enhanced in co-cultivation with hydrogenotrophic methanogens³⁸ which was also increased in abundance with the increasing effluent recirculation rates. Particularly, genus Methanothrix had increased in abundance at R of 1.5, reaching 17.1%. The same conclusion was applied to the Synergistes and the Syntrophomonas. The unsaturated Long-chain fatty acids (LCFA) can be utilized by the Syntrophomonas³⁹ and the “Synergistes” group of organisms are a phylogenetic cluster of Gram-negative anaerobes related to Synergistes jonesii, sufficiently different from all other phyla and to be considered a distinct phylum. They are widely distributed in nature although normally only a minor constituent of the bacterial community in each habitat. They have evolved to adapt to each habitat, and therefore exhibit a wide range of physiological and biochemical characteristics.⁴⁰ Moreover, the Synergistes which is an acetate-utilizing bacteria in the anaerobic digester sludge⁴¹ plays an important role in degradation of phenol and facilitating the conversion of phenol to benzoate. As the first step in anaerobic degradation of phenol pathway is the conversion of phenol to benzoate, the benzoate is then dearomatized to form cyclohexane carboxylic acid, the ring structure of which is then cleaved to form heptanoate. Heptanoate is degraded to form acetate (methane precursor). The conversion of phenol to benzoate was demonstrated to be the rate-limiting step in the anaerobic degradation of phenol.⁴² Although, the relative abundance of these genera was increased with increasing effluent recirculation rates, it did not show a good performance on phenol removal and methane yield. The effluent total phenol increased from 70 mg L⁻¹ in R = 0.5 to 110 mg L⁻¹ in R = 1. With respect to the methane production rate decreased from 300 (mL CH₄ per g per d) in R = 0.5 to 100 (mL CH₄ per g per d) in R = 1. The loss of the biomass in the system resulted in the dramatically dropped removal efficiency of COD, total phenol and low methane production because of excessive effluent recirculation.

The proportions of Bacteroidetes and Firmicutes were remained at a certain level. Many species belonging to Bacteroidetes and Firmicutes are hydrolytic fermenting bacteria or acidogens. As shown in the Fig. 6a, the growths and the survivals of some species belonging to Bacteroidetes and Firmicutes were not inhibited in the CGW. Even they can be reproduced due to stimulation of the moderate up-flow velocity. When the effluent recirculation rate was increased to 0.5, the corresponding proportion reached as high as 20.67% and 17.2% respectively in the sequence analysis result. The removal rates of COD and total phenol rose to around 70% and 65%, respectively, the methane production rate was increasing to 300 mL CH₄ per g per d.

As shown in Fig. 6a, when R = 0, apparently the Proteobacteria increased from 12.4% in the seed to 39.4% and it would be expected to be predominant in AF. Many species belonging to Proteobacteria are acetic acid bacteria,⁴³ which has strong tolerance to toxic substances. Moreover, when R = 0, they have high abundance and exhibited good performance in the degradation of toxic pollutants in CGW. Also, the effluent total phenol was decreased to 100 mg L⁻¹.

When R = 1.5, the relative abundance of the Proteobacteria was decreased to 12.2%. The bacteria from Spirochaetes, typically, have a helical coiled morphology and they grow chemoheterotrophically. It was reported that they have metabolic activities including acetate, ethanol, and lactate fermentations from glucose,³⁹ as well as acetate oxidation.⁴⁴ In the system, it was decreased from 5.5% (R = 0) to 0.8% (R = 1.5). Additionally, the Geobacter, Syntrophorhabdus, Treponema, and so on displayed the decreasing trend in abundance when the effluent recirculation rate was increased. However, the reduction in abundance with the increasing effluent recirculation rates likely lead to the unsatisfied performance in lab-scale operation to some extent. Lab-scale research also showed that when R = 1.5, the effluent COD and total phenol were sharply fluctuated and the removal rates of COD and total phenol even decreased to 30% and 20%, respectively. The methane production rate reached 0.032 mL CH₄ per g per d.

4. Conclusion

By kinetic equation analysis, the optimum ratio of PSW and CGW was 1 : 1 in influent. Adding PSW as co-substrate can significantly enhance the treatment efficiency, but extending HRT to 64 h, there is no further obvious improvement in toxic removal and methane yield. Adopted suitable effluent recirculation on co-digestion could result in high activity of some anaerobic bacteria. The effluent total phenol reached 70 mg L⁻¹ at effluent recirculation rate of 0.5 : 1. The analysis of microbial community composition in anaerobic sludge under different recirculation rates demonstrated the phenol degradation and methane yield had some relationship with the main microbial communities.

Acknowledgements

The authors express their gratitude to the School of Environmental Science and Engineering, State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University for providing the research facilities and the guide of Prof. Fan Lü from State Key Laboratory of Pollution Control and Resources Reuse, Tongji University, Shanghai, China.

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