Abstract

Trace elements play a critical role for microbial activity in anaerobic digestion (AD) but their effects were probably overestimated in batch tests and should be comparably evaluated in continuous systems. In this study, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+ were added in different concentrations to manure wastewater, and the effects were compared in both batch and continuous systems. The results were used to demonstrate suitable trace element compositions for AD of dairy and swine wastewater, and to compare the outcomes from batch and continuous systems. Fe2+ and Zn2+ were identified as being the most efficient stimulant of dairy and swine wastewater respectively. The addition of 5 mg/L Fe2+ and 0.4 mg/L Zn2+ increased the batch specific methane yield by 62% and 126% for dairy and swine wastewater, respectively. Nevertheless, a lower increment of 2% and 21%, for dairy and swine wastewater was obtained in the 120-day continuously-fed experiments. The 16S rRNA gene sequencing results indicated a relationship between the methanogens population, specific methanogenic activities, propionate, and dissolved hydrogen. Conclusively, the addition of a low dosage of Fe2+ and Zn2+ is a feasible strategy to enhance the methanogenic metabolism of the AD of dairy and swine wastewater respectively.

INTRODUCTION

In China, around 4 billion tons of animal manure and wastewater is produced annually (Jiang et al. 2011). The quantity of cattle and swine manure and wastewater is proportionally the largest at 3.2 billion tons, accounting for approximately 80% of the animal manure generated (Jiang et al. 2011). Anaerobic digestion (AD) is considered to be one of the most effective techniques for treating and disposing of manure wastewater in order to mitigate the discharge of chemical oxygen demand (COD) whilst also simultaneously generating clean energy as bio-methane, and producing organic fertilizer as digestate. However, the methane yield from the AD of animal manure is often relatively low and there is a need to find more advanced approaches to increase the efficiency of anaerobic degradation (Qiao et al. 2011). The addition of trace elements has been shown to increase methane yield, enhance the degradation efficiency, and alleviate the accumulation of volatile fatty acids (VFAs) in various types of substrate, such as food waste (Westerholm et al. 2015), crop residues (Lebuhn et al. 2008) and stillage (Gustavsson et al. 2011).

Typically, iron, nickel, cobalt, copper and zinc are essential elements for methanogenic enzymes and co-factors. The trace elements were reported to play a key role as co-factors in enzymes for enhancing the methanogenesis (Kida et al. 2001). Trace elements can strongly bind to sulfur and thus mitigate the negative effects of sulfide. Recently, the addition of Fe2+ and Ni2+ was found to alter the methanogenic pathway and subsequently increase the microbial activity under an ammonia-stressed environment (Bi et al. 2019). On the other hand, the anaerobic system was normally operated in a continuously fed mode instead of a batch feeding pattern. However, the different effects of the addition of trace elements in batch and continuous AD of manure wastewater as the sole substrate have been infrequently studied. A methane yield enhancement of as much as 55% for chicken manure was reported in a batch experiment by Zhang et al. (2012), but a lower enhancement; that is, 34%, was found in a recent study (Bi et al. 2019). That may indicate the effects of trace elements were most probably overestimated and should be reevaluated in continuous systems. So far, whether the addition of trace metals can have comparable enhancement effects in batch and continuous anaerobic system is still unclear. On the other hand, although the deficiency of trace elements in the anaerobic system has been widely reported to induce low methane yield and process instability, an overdose could have toxicity effects on the microorganisms (Thanh et al. 2016). At the same time, a high dosage of trace elements for enhancing methane production has also been reported; that is, 125 mg/L Zn2+ for swine manure (Zhang et al. 2017) and 100–500 mg/L Fe2+ for chicken manure (Zhang et al. 2012). The significant addition of trace elements increases the cost of a biogas plant's operation and may induce excess metals to enter the environment. Consequently, the appropriate dosage used for the trace elements supplements for AD of animal manure and wastewater is a matter that is yet to be answered.

In an anaerobic digestion system, the methanogens utilize acetate or H2/CO2 to produce methane. The addition of trace elements thus enhances the activities of methanogens, resulting in an increase in the methane yield. AD is an intricate multi-stage process reliant on the activities of diverse microbial communities for hydrolysis, acidogenesis and methanogenesis. However, there are few studies that have investigated the effects of trace elements on both acetoclastic and hydrogenotrophic methanogenic activities.

This study, therefore, aimed to establish the suitability and appropriate dosage of the trace elements as well as explaining the key role that single trace elements play in a batch experiment. The effects of the trace elements on enhancing the methane yield was further investigated in long term continuously operated digesters, and the methanogenesis activities and the methanogenic communities were analyzed to discover the microbial dynamics with the addition of trace elements.

MATERIALS AND METHODS

Characteristics of wastewater and inoculums

The dairy wastewater was collected from a dairy farm located in Beijing. The sampled wastewater was blended into homogenous slurry in the laboratory by using a blender (Joyoung JYLC012, Jinan) for 5 minutes. The inoculum used in this study was taken from a biogas plant treating dairy manure under mesophilic conditions. The swine wastewater was collected from a swine breeding farm located in Beijing. The inoculum used in the swine wastewater experiment was obtained from a CSTR at this farm. The two digesters were maintained at mesophilic conditions. The diary wastewater had a mean concentration of total solids (TS) and volatile solids (VS) of 44.8 and 35.3 g/L. The TS and VS of the swine wastewater was 8.4 and 4.5 g/L. The inoculum for the dairy and swine wastewater reactor had a TS of 30.8 and 5.8 g/L respectively. The Fe2+, Co2+, Ni2+, Cu2+ and Zn2+ in the dairy wastewater was 0.865, 0.022, 0.041, 0.024 and 0.158 mg/L. Their concentration in swine wastewater was 0.985, 0.109, 0.062, 0.098 and 0.455 mg/L. The characteristics of wastewater and inoculums are provided in Table S1 (supplementary materials).

Batch experiments

Batch anaerobic digestion experiments (30 days) were carried out by conducting biochemical methane potential tests according to a previous study (Wandera et al. 2018). The batch experiments were carried out in duplicate at 37 °C using 120 mL serum bottles into which 70 mL of inoculums and 10 mL of dairy wastewater was added. For the swine wastewater experiment, 50 mL of inoculums and 30 mL of swine wastewater were added. The dairy manure and swine wastewater had a VS concentration of 34.5 g/L and 4.5 g/L respectively. In each batch experiment, one of the following trace elements was added to reach different concentrations in the media: Fe2+ (5, 10, 20 mg/L), Co2+ (0.1, 0.2, 0.4 mg/L), Ni2+ (0.1, 0.2, 0.4 mg/L), Cu2+ (0.1, 0.2, 0.4 mg/L), Zn2+ (0.1, 0.2, 0.4 mg/L). The dosage was chosen based on previously reported values (Zandvoort et al. 2006a, 2006b; Qiao et al. 2011; Qiang et al. 2012; Voelklein et al. 2017). Two control bottles filled with inoculums but without substrates and trace elements were incubated in parallel. The gas volume produced from batch bottles was measured using a syringe. The methane content of the biogas was analyzed by using a gas chromatograph (GC-8A, Shimadzu, Japan). The methane production potential was obtained by simulating the gas production using the widely used modified Gompertz model (Wandera et al. 2018; Zhao et al. 2018) and is provided in the supplementary materials.

Continuously stirred tank reactors

Four glass bottles with a total volume of 2 L (working volume, 1.6 L) were used as the continuously stirred tank reactors (CSTRs). Two of them were used as control. The CSTRs operated in parallel for 120 days and were maintained at 37 ± 1 °C using a water bath (HH-60, ChangzhouGuohua, China). The hydraulic retention time (HRT) of the dairy manure digesters was set at 16 days and the organic loading rate (OLR) was fixed at 2.21 kgVS/m3/d. One reactor received dairy wastewater mixed with Fe2+ to reach a final concentration of 5 mg/L. One parallel CSTR treating dairy wastewater without Fe2+ addition operated as control. A similar setup was established for the swine wastewater, in which one CSTR received substrate with 0.4 mg/L Zn2+ and one control CSTR was fed only substrate. The HRT and OLR of the swine wastewater CSTRs were set at 10 days and 0.45 kgVS/m3/day, respectively. The flow rate of the dairy and swine manure digester were 1.0 and 1.6 L/d respectively. The feeding was conducted manually once per day. The setup of the CSTR system is shown in Figure 1.

Chemical analysis

TS, VS, COD, VFA, NH4+-N, and alkalinity were measured according to a previous study (Bi et al. 2019). The soluble trace elements in the liquid phase of digestate were analyzed by an inductively coupled plasma-optical emission spectroscopy (Optima 7300DV, USA). The analytical wavelengths (nm) were chosen as follows: Fe (238.204), Co (228.616), Ni (231.604), Cu (327.393), and Zn (206.200). The effluents from the batch serum was centrifuged for 20 minutes at a speed of 10,000 r/min to obtain supernatants. The supernatants were obtained by passing the liquid through membrane filter (pore size, 0.45 μm). The pH of filtrate was adjusted to approximately 2.0 with HNO3 for 30 minutes. The acidified liquid was further filtered by 0.45 μm membrane filters and then the filtrate was analyzed for concentrations of metal.

Acetoclastic methanogenic activity test

The specific methanogenic activity (SMA) of the CSTR were measured by using NaCH3COO (COD 2,000 mg/L) and NaCH3CH2COO (COD 500 mg/L) as the methanogens' substrates. The SMA was obtained by dividing the slope of the linear section of the accumulated methane production rate with the mass of volatile solid weight (Bi et al. 2019). The volume of the biogas and methane content was measured at an interval of two hours in the batch experiments.

Hydrogenotrophic methanogenic activity test

The consumption rate of the dissolved hydrogen by hydrogenotrophic methanogens was tested through incubating batch bottles in a water bath measuring 37 ± 1 °C. Two glass bottles (100 mL) were used in the experiment. The first bottle was filled with 70 mL of fresh effluent from the digesters and 70 mL of hydrogen saturated water (37 °C). It was subsequently sealed with air tight cellotape. The second bottle (control) was filled with 70 mL of distilled water and 70 mL of hydrogen saturated water. The consumption rate of the dissolved hydrogen in the two bottles was determined by two H2-100 microsensors under +1,000 mV connected to an in-situ microsensor (Unisense, Denmark). The hydrogen microsensors were pre-polarized by using distilled water under +1,000 mV for 24 h and then was calibrated by water saturated with hydrogen (Zhao et al. 2018). Duplicate experiments were carried out for measuring the hydrogenotrophic methanogenic activities.

The first order kinetic model was used to simulate the consumption rate of dissolved hydrogen.
formula
where CSo is the maximum gas produced (mL); Cs is the maximum gas production minus the accumulated gas (mL); k is rate constant (h−1); t is the time (h).

The analysis of microbial community

Sludge samples were withdrawn from the continuously operating digesters after 120 days of operation and stored at −20 °C until analysis. The CTAB/SDS method was used for total genomic DNA extraction (Chen et al. 2010). The polymerase chain reaction (PCR) amplifications were implemented by using the primer 338F/806R for bacterial and 524F/958R for archaeal sequences. The thermal cycling process was performed according to a previous research (Wandera et al. 2018). The mixture PCR products were purified by using the Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using the NEB Next® Ultra™ DNA Library PrepKit for Illumina (NEB, USA). The library quality was assessed on the Qubit@ 2.0Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina HiSeq platform, and 250 bp paired-end reads were generated. The paired-end reads from the original DNA fragments were merged using FLASH (Magoc & Salzberg 2011) and was analyzed according to a previous research (Caporaso et al. 2011). Finally, the sequences were clustered into operational taxonomic units (OTUs) (at 97% similarity). Typical sequences were chosen for each OTU. Taxonomic classification was performed with 16S rRNA reference (RDP) database. The detailed analysis of microbial community methods were introduced in a previous study (Jiang et al. 2019) and added in the supplementary methods.

RESULTS AND DISCUSSION

Effects of trace elements on batch methane production potential

The addition of trace elements enhanced the methane yield from dairy manure waste water in the 30-day batch experiments (Table 1). Fe2+ had the most significant effect and the addition of 5, 10 and 20 mg/L increased the methane yield by 62%, 20% and 14%, respectively (i.e. from 351 to 570, 422 and 401 mL/g-VSin). For the other metals, the highest increase in the methane yield was 31% for 0.4 mg/L Zn2+, 25% for 0.4 mg/L Ni2+, 23% for 01 mg/L Co2+, and 9% for 0.1 mg/L Cu2+. It was supposed that the addition of trace elements enhances the binding with sulfur and, thus, has a positive effect on the AD process. Several other studies have shown positive effects by the addition of Fe salts to manure containing materials (Zhang et al. 2016). In the current study, the results indicated that the Fe2+ played a critical role in the rise of the methane yield in dairy wastewater. At the same time, the maximum methane production rate (Rmax) also slightly increased with the addition of each metal (Table 1).

Table 1

Methane production potential of dairy wastewater in batch experiments with and without the addition of trace elements

Dairy wastewater
Swine wastewater
Added trace elements (mg/L)P0 (mL-CH4/gVSin)P0 increase (%)Rmax (mL-CH4/gVSin/d)λ(d)P0 (mL-CH4/gVSin)P0 increase (%)Rmax (mL-CH4/gVSin/d)λ(d)
Control 351 28 <0.1 1,016 70 5.8 
Fe2+ 570 62 31 <0.1 1,058 65 5.5 
10 422 20 30 <0.1 1,174 16 87 2.3 
20 401 14 31 <0.1 1,430 41 70 4.7 
Co2+ 0.1 430 22 32 <0.1 1,032 77 2.6 
0.2 413 18 32 <0.1 1,029 75 2.1 
0.4 404 15 31 <0.1 901 11 69 2.5 
Ni2+ 0.1 385 10 31 <0.1 1,096 80 2.5 
0.2 411 17 30 <0.1 1,075 74 2.4 
0.4 438 25 33 <0.1 931 −8 40 2.3 
Cu2+ 0.1 383 28 <0.1 978 −4 70 
0.2 378 31 <0.1 1,030 74 
0.4 338 −4 27 <0.1 1,414 39 100 
Zn2+ 0.1 364 30 <0.1 1,300 28 92 2.6 
0.2 381 30 <0.1 1,782 75 116 3.1 
0.4 458 31 34 <0.1 2,300 126 141 4.3 
Dairy wastewater
Swine wastewater
Added trace elements (mg/L)P0 (mL-CH4/gVSin)P0 increase (%)Rmax (mL-CH4/gVSin/d)λ(d)P0 (mL-CH4/gVSin)P0 increase (%)Rmax (mL-CH4/gVSin/d)λ(d)
Control 351 28 <0.1 1,016 70 5.8 
Fe2+ 570 62 31 <0.1 1,058 65 5.5 
10 422 20 30 <0.1 1,174 16 87 2.3 
20 401 14 31 <0.1 1,430 41 70 4.7 
Co2+ 0.1 430 22 32 <0.1 1,032 77 2.6 
0.2 413 18 32 <0.1 1,029 75 2.1 
0.4 404 15 31 <0.1 901 11 69 2.5 
Ni2+ 0.1 385 10 31 <0.1 1,096 80 2.5 
0.2 411 17 30 <0.1 1,075 74 2.4 
0.4 438 25 33 <0.1 931 −8 40 2.3 
Cu2+ 0.1 383 28 <0.1 978 −4 70 
0.2 378 31 <0.1 1,030 74 
0.4 338 −4 27 <0.1 1,414 39 100 
Zn2+ 0.1 364 30 <0.1 1,300 28 92 2.6 
0.2 381 30 <0.1 1,782 75 116 3.1 
0.4 458 31 34 <0.1 2,300 126 141 4.3 

For swine wastewater, a higher methane yield (1,016 mL/gVSin) than that from dairy manure was obtained in the batch experiments (Table 1). At the same time, a significant increment in the methane yield was observed when the elements were added. For example, the addition of 0.2 and 0.4 mg/L Zn2+ increased the methane yield by 75% and 126%, respectively. The present study thus shows that even a lower dosage of 0.2–0.4 mg/L Zn2+ has comparable and positive effects on the methane yield increment. A significant increase in the methane production rate was also observed with the addition of Zn2+; that is, from 70 to 141 ml/gVS/d (0.4 mg/L Zn2+). The addition of Co2+ and Ni2+ did not significantly increase the methane yield from swine manure. The highest positive effect on methane yield was obtained from the addition of 0.4 mg/L Cu2+ (+39%) and 20 mg/L Fe2+ (+41%) in Table 1. This result indicates that both Fe2+ and Zn2+ have a critical role in the enhancement of the methane yield from dairy and swine wastewater, and that even a lower dosage than that used in previously reported research (Zhang et al. 2014, 2016) can play a positive role.

Effects of trace elements on long term CSTR reactors

The batch experiments results indicated that the Fe2+ (5 mg/L) had the most significant effect on dairy wastewater, and Zn2+ addition was most beneficial in swine wastewater. Consequently, this was further investigated in continuously operating systems. As shown in Figure 2(a), Fe2+ addition in the dairy wastewater reactor increased the methane yields by 12% (84 mL/g-VSin) compared with the control (75 mL/g-VSin) with a P = 0.033. At the same time, the concentration of VFAs were lower in dairy wastewater after the addition of Fe2+ compared to the control digester with a P < 0.05. The VFA concentration was shown in Figure 2(c). From the 43th day to the end of the experiment, the acetic acids in the Fe2+ added digester were significantly (P < 0.05) lower than in the control one. However, compared to the results obtained in the batch experiments, the enhancement of the methane yield in the continuous experiment with Fe2+ was much lower.

Figure 1

The setup of the CSTR system.

Figure 1

The setup of the CSTR system.

Figure 2

Methane yield and VFA variations in the digesters with and without the addition of metals.

Figure 2

Methane yield and VFA variations in the digesters with and without the addition of metals.

The summary of the long term continuously-fed reactors performance was provided in Table 2. The swine wastewater continuous digester supplemented with 0.4 mg/L Zn2+ had a 21% higher methane production compared to the control (192 in control and 238 mL/g-VSin in Zn2+ supplemented reactor). The increment in the continuous experiment was also much lower than that obtained in the batch experiment. A longer time is therefore required to obtain high methane production even with the addition of trace metals. The total concentration of VFAs in the swine wastewater digesters was 192 mg/L without Zn2+ and 66 mg/L with Zn2+. The period between the 85th and 122nd day indicated a stable but low VFA concentration, i.e. 53 mg/L (without Zn2+) and 48 mg/L (with Zn2+). As previously reported, the VFA reduced from 2,100 mg/L to 400 mg/L when trace elements (Fe, Co, Ni, Se, and W) were added to the mixture of manure and industrial waste (Qiao et al. 2011), suggesting that the methanogenic activity in the swine wastewater digester may be enhanced by adding the trace elements. The VFA concentration in swine digesters was shown in Figure 2(e) and 2(f).

Table 2

Summary of long term digester performance

ParametersUnitsDairy wastewaterDairy wastewater + 5 mg/L Fe2+Swine wastewaterSwine wastewater + 0.4 mg/L Zn2+
HRT days 16 16 10 10 
OLR g-VS/(L·d) 2.2 2.2 0.45 0.45 
pH 7.96 ± 0.20 7.98 ± 0.20 7.91 ± 0.18 7.86 ± 0.20 
Gas production mL/(L·d) 203 ± 20 222 ± 7 102 ± 4 127 ± 3 
CH4 64.7 ± 5.4 67.6 ± 2.5 67.7 ± 2.5 67.1 ± 2.4 
CO2 35.1 ± 4.4 31.6 ± 3.3 33 ± 0.03 32.1 ± 3.8 
CH4 yield mL/g-VS 74 ± 9 85 ± 3 192 ± 10 238 ± 9 
Acetate mg/L 79 ± 4 39 ± 14 80 ± 3 59 ± 5 
Propionate mg/L 94 ± 7 109 ± 7 22 ± 7 
Butyrate mg/L 
VFAs mg/L 125 ± 4 48 ± 6 192 ± 8 66 ± 5 
ParametersUnitsDairy wastewaterDairy wastewater + 5 mg/L Fe2+Swine wastewaterSwine wastewater + 0.4 mg/L Zn2+
HRT days 16 16 10 10 
OLR g-VS/(L·d) 2.2 2.2 0.45 0.45 
pH 7.96 ± 0.20 7.98 ± 0.20 7.91 ± 0.18 7.86 ± 0.20 
Gas production mL/(L·d) 203 ± 20 222 ± 7 102 ± 4 127 ± 3 
CH4 64.7 ± 5.4 67.6 ± 2.5 67.7 ± 2.5 67.1 ± 2.4 
CO2 35.1 ± 4.4 31.6 ± 3.3 33 ± 0.03 32.1 ± 3.8 
CH4 yield mL/g-VS 74 ± 9 85 ± 3 192 ± 10 238 ± 9 
Acetate mg/L 79 ± 4 39 ± 14 80 ± 3 59 ± 5 
Propionate mg/L 94 ± 7 109 ± 7 22 ± 7 
Butyrate mg/L 
VFAs mg/L 125 ± 4 48 ± 6 192 ± 8 66 ± 5 

In this study, the continuous digester treating dairy and swine wastewater was operated for 120 days with HRTs of 8 and 12 for the digesters. The enhanced performance obtained from the long term experiment was therefore representative. However, by comparing the batch experiments, the significantly enhanced methane production through the addition of trace metals did not happen in the long term experiment. From this point of view, the evaluation of the effects of trace metals on methane production should be investigated by operating long term continuous digesters to obtain more useful and reliable results.

It was reported that in a neutral pH environment, approximately 50% of the hydrogen sulfide was available as bio-sulfide ion HS to potentially precipitate trace elements (Voelklein et al. 2017). The precipitation of FeS and the co-precipitation of S with Co, Ni, Zn and Cu in an anaerobic system would affect the uptake of the metals by microorganisms. The Fe was more easily precipitated to FeS and this thus minimized the formation of H2S (Wei et al. 2018). In the current study, the addition of Fe was also higher than for other elements. In light of the observations in the current study, the different roles of Fe2+ and Zn2+ on dairy and swine wastewater still need further investigation. On the other hand, the effects of trace elements addition in continuous AD was much lower than that obtained in batch AD. The may indicate the evaluation of trace elements effects using batch experiment procedure may overestimated the effects.

Effects of trace elements on microbial communities

The analysis of the microbial community showed that the Clostridia and Bacteroidia were the dominant bacterial classes in the continuously operating digesters after 120 days of operation (Table 3). In the swine wastewater digester, the addition of 0.4 mg/L Zn2+ slightly decreased the class Clostridia from 43.8% to 39.2%. At class level, the methanogenic communities were clearly dominated by Methanomicrobia, which represented 88–92% of the total archaeal community in both digesters. The addition of the metals only had minor effects on the methanogens population. Compared to the cow wastewater (2.8–3.2%) reactors, the Methanobacteria in swine wastewater (with and without Zn2+ addition) was significantly higher, i.e. 11–12%. In cow wastewater digesters, the methylotrophic methanogen Thermoplasmata (Poulsen et al. 2013) was also found at 5.2–5.5%; however, in the swine wastewater digesters, the Thermoplasmata was less than 1% (Table 3).

Table 3

Summary of bacterial and archaeal community under class level, %

Cow wastewaterCow wastewater (+ 5 mg/L Fe)Swine wastewaterSwine wastewater (+ 0.4 mg/L Zn)
C_Bacterial Clostridia 35.2 29.5 43.8 39.2 
Bacteroidia 28.1 31.9 14.9 15.2 
Synergistia 1.8 4.1 0.7 1.3 
Spirochaetes 3.1 4.5 3.5 4.4 
Gammaproteobacteria 5.6 2.8 9.5 12.3 
Fibrobacteria 3.6 2.9   
Erysipelotrichia 4.6 2.9 1.3 1.0 
Epsilonproteobacteria 3.1 2.8 13.2 16.3 
Deltaproteobacteria 2.3 2.1 1.5 1.3 
Betaproteobacteria 1.7 1.3 4.7 3.3 
Bacteroidetes 2.4 2.2 
Anaerolineae 2.6 4.8 
Cloacimonetes 1.8 1.1 
Others 5.9 8.1 5.1 4.5 
C_Archaeal Methanomicrobia 91.6 91.7 87.9 88.3 
Methanobacteria 3.2 2.8 11.7 11.1 
Thermoplasmata 5.2 5.5 
Others 0.4 0.6 
Cow wastewaterCow wastewater (+ 5 mg/L Fe)Swine wastewaterSwine wastewater (+ 0.4 mg/L Zn)
C_Bacterial Clostridia 35.2 29.5 43.8 39.2 
Bacteroidia 28.1 31.9 14.9 15.2 
Synergistia 1.8 4.1 0.7 1.3 
Spirochaetes 3.1 4.5 3.5 4.4 
Gammaproteobacteria 5.6 2.8 9.5 12.3 
Fibrobacteria 3.6 2.9   
Erysipelotrichia 4.6 2.9 1.3 1.0 
Epsilonproteobacteria 3.1 2.8 13.2 16.3 
Deltaproteobacteria 2.3 2.1 1.5 1.3 
Betaproteobacteria 1.7 1.3 4.7 3.3 
Bacteroidetes 2.4 2.2 
Anaerolineae 2.6 4.8 
Cloacimonetes 1.8 1.1 
Others 5.9 8.1 5.1 4.5 
C_Archaeal Methanomicrobia 91.6 91.7 87.9 88.3 
Methanobacteria 3.2 2.8 11.7 11.1 
Thermoplasmata 5.2 5.5 
Others 0.4 0.6 

Note: the ‘others’ were less than <1%.

The analysis of the methanogenic communities showed different structures at genus level in diary and swine manure digesters, whereas the addition of Fe2+ or Zn2+ had a minor effect on the community composition (Figure 3). In dairy wastewater digesters, the obligate acetoclastic methanogen Methanosaeta represented 35–40% of the total archaeal community both without and with Fe2+ (Figure 3(a)). Similarly, the relative abundance of Methanosarcina slightly decreased from 48% to 46% with Fe2+ addition. Methanosaeta has been shown to use acetate at a low concentration with a minimum threshold of acetate at 7–70 μmol/L, while Methanosarcina required higher concentrations of acetate in the range of 200–1,200 μmol/L (Jetten et al. 1992). In the current study, the slight decrease in acetic acid concentration from 91 to 31 mg/L and the enhanced methane yield in the digester with the addition of Fe2+ indicate that this addition of metals may improve the growth and activities of acetoclastic methanogens.

Figure 3

Methanogenic communities and acetic acid in digesters with and without the addition of metals. (a) Dairy wastewater digester; (b) swine wastewater digester.

Figure 3

Methanogenic communities and acetic acid in digesters with and without the addition of metals. (a) Dairy wastewater digester; (b) swine wastewater digester.

In the swine wastewater digesters, Methanosaeta was also the dominant methanogen at a total relative abundance of 42–44%, both with and without the addition of Zn2+. The relative abundance of hydrogenotrophic methanogens (including Methanocorpusculum, Methanobrevibacter, Methanoculleus, Methanospirillum, and Methanosphaera) was of a similar number of around 37% in the swine wastewater digester, irrespective of the Zn2+ addition. Methanocorpusculum showed a high percentage in the swine wastewater digester with and without Zn2+ addition, and the addition of Zn2+ slightly increased from 15.5% to 16.6%. Methanobrevibacter accounted for 5.9% and 5.2% for the swine wastewater digester with and without Zn2+ addition. Methanocorpusculum and Methanobrevibacter may have played an important role in the anaerobic digestion of swine wastewater, since those methanogens were previously found in swine waste storage pits (Whitehead & Cotta 1999); however, in the dairy wastewater digester, those two methanogens were not observed.

Effects of trace elements on methanogenic activities

The specific methanogenic activity (SMA) of the dairy wastewater digester showed a significantly increased methane production rate from 0.245 to 0.367 mL-CH4/gVS/h from acetate when Fe2+ was added to the digester (Figure 4). The significant increase in SMA is in line with previous studies showing a 43.1% increase in SMA with trace elements supplementation (iron, nickel, cobalt, and zinc) in an anaerobic membrane bioreactor treating food processing wastewater (Yu et al. 2016) and a 74.8% increase with the addition of Co2+ (5 μmol/L) (Zandvoort et al. 2006a, 2006b). The propionic acid was a major intermediate and important metabolite in the anaerobic process. In the current study, the positive effects of trace elements on propionic acid degradation were also observed. The addition of Fe2+ in the digester treating dairy wastewater significantly enhanced the SMA of propionic acid from 0.0469 to 0.0642 mL-CH4/gVS/h (Figure 4). A high SMA of propionic acid from 0.6875 to 1.0395 mL-CH4/gVS/h was found in the swine wastewater digesters, with Zn2+ addition increasing it significantly by 51.2%.

Figure 4

Specific methanogenic activity of acetate and propionate in dairy and swine wastewater digesters.

Figure 4

Specific methanogenic activity of acetate and propionate in dairy and swine wastewater digesters.

During the anaerobic digestion process, dissolved hydrogen was an important precursor for methane formation. In the current study, the hydrogenotrophic methanogenesis activity was investigated by testing the dissolved hydrogen concentration in batch experiments. A higher consumption rate (P = 0.0455) was found in the dairy wastewater feeding digester with Fe2+ addition (Figure 5(a) and 5(b)). The hydrogen consumption rate of dissolved hydrogen increased from 0.1155 h−1 in the controlled dairy wastewater digester to 0.1325 h−1 in the Fe2+ supplemented digester, indicating that Fe2+ addition induced the development of archaeal communities with higher activity. Investigation of swine wastewater digesters indicated the SMA of acetic acid was 9.024 and 5.394 mL-CH4/gVSin/h with and without Zn2+ supplement. An increment of SMA by 67.3% was obtained through the addition of 0.4 mg/L Zn2+. However, the dissolved hydrogen consumption rate of swine wastewater was not significantly increased with a P value of 0.11 when Zn2+ was added (Figure 5(c) and 5(d)). As the hydrogenotrophic methanogens in swine wastewater with and without metals addition were higher than those in dairy wastewater, it was therefore supposed that the swine wastewater already had higher capacities for hydrogen consumption. Consequently, the addition of Zn2+ did not significantly increase the hydrogen consumption rate in swine wastewater. The results showed that the increment in the methane production yield when trace elements were added in the dairy and swine manure wastewater anaerobic digesters could be induced by the improved methanogenic activities, which also enhanced the conversion of both acetate and propionate and lowered the level of dissolved hydrogen.

Figure 5

Hydrogen consumption rate of dairy and swine wastewater digesters.

Figure 5

Hydrogen consumption rate of dairy and swine wastewater digesters.

CONCLUSION

The effects of each metal on enhancing the methane yield of dairy and swine wastewater were obtained through the batch experiments. The key roles of Fe2+ and Zn2+ were identified in the anaerobic digestion of dairy and swine wastewater. The methane yield in the long-term continuous experiment was also increased by adding a low concentration of metals but the degree was much lower than those obtained in the batch tests. The addition of Fe2+ and Zn2+ also induced the changes in the methanogen populations, which may explain the increased consumption rate of the dissolved hydrogen, and the specific methanogenic activities.

Supplementary data for this work can be found in the electronic version of this paper (Figure S1, methane production potential of dairy wastewater with and without the addition of metals; Figure S2, methane production potential of swine wastewater with and without the addition of metals; Figure S3, bacterial community at day 120 of operation with and without the addition of elements; Table S1, Characteristics of inoculum and dairy and swine wastewater used in batch and continuous experiments; Table S2, Summary of bacterial and archaeal community under class level, %; Equation S1, modified Gompertz model; Method S1, the analysis of microbial community).

ACKNOWLEDGEMENTS

This work was partially supported by the National Key Research and Development Program of China (2016YFD0501403) and Beijing Municipal Natural Science Foundation (No. 6182017).

SUPPLEMENTARY MATERIAL

The Supplementary Material for this paper is available online at https://dx.doi.org/10.2166/wst.2019.420.

REFERENCES

Caporaso
J. G.
Lauber
C. L.
Walters
W. A.
Berg-Lyons
D.
Lozupone
C. A.
Turnbaugh
P. J.
Fierer
N.
Knight
R.
2011
Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample
.
Proc. Natl. Acad. Sci. USA
108
,
4516
4522
.
Chen
H.
Rangasamy
M.
Tan
S. Y.
Wang
H.
Siegfried
B. D.
2010
Evaluation of five methods for total DNA extraction from western corn rootworm beetles
.
PLoS One
5
,
e11963
.
Jiang
X. Y.
Sommer
S. G.
Christensen
K. V.
2011
A review of the biogas industry in China
.
Energ. Policy
39
,
6073
6081
.
Jiang
M. M.
Qiao
W.
Ren
Z. R.
Mahdy
A.
Wandera
S. M.
Li
Y. Y.
Dong
R. J.
2019
Influence of operation conditions on methane production from swine wastewater treated by a self-agitation anaerobic reactor
.
Internat. Biodeter. Biodegr
.
143
,
104710
.
Poulsen
M.
Schwab
C.
Borg Jensen
B.
Engberg
R. M.
Spang
A.
Canibe
N.
Højberg
O.
Milinovich
G.
Fragner
L.
Schleper
C.
Weckwerth
W.
Lund
P.
Schramm
A.
Urich
T.
2013
Methylotrophic methanogenic thermoplasmata implicated in reduced methane emissions from bovine rumen
.
Nat. Commun.
4
,
66
78
.
Thanh
P. M.
Ketheesan
B.
Yan
Z.
Stuckey
D.
2016
Trace metal speciation and bioavailability in anaerobic digestion: a review
.
Biotechnol. Adv.
34
,
122
136
.
Wandera
S. M.
Qiao
W.
Algapani
D. E.
Bi
S.
Yin
D.
Qi
X.
Liu
Y.
Dach
J.
Dong
R.
2018
Searching for possibilities to improve the performance of full scale agricultural biogas plants
.
Renew. Energ.
116
,
720
727
.
Wei
J.
Hao
X.
Loosdrecht
M. C. M.
Li
J.
2018
Feasibility analysis of anaerobic digestion of excess sludge enhanced by iron: a review
.
Renew. Sust. Energ. Rev.
89
,
16
26
.
Whitehead
T. R.
Cotta
M. A.
1999
Phylogenetic diversity of methanogenic archaea in swine waste storage pits
.
FEMS Microbiol. Lett.
179
,
223
226
.
Zandvoort
M. H.
Hullebusch
E. D. V.
Gieteling
J.
Lens
P. N. L.
2006a
Granular sludge in full-scale anaerobic bioreactors: trace element content and deficiencies
.
Enzyme Microb. Tech.
39
,
337
346
.
Zandvoort
M. H.
van Hullebusch
E. D.
Fermoso
F. G.
Lens
P. N. L.
2006b
Trace metals in anaerobic granular sludge reactors: bioavailability and dosing strategies
.
Eng. Life Sci.
6
,
293
301
.
Zhang
W.
Guo
J.
Wu
S.
Dong
R.
Zhou
J.
Lang
Q.
Li
X.
Lv
T.
Pang
C.
Chen
L.
Wang
B.
2012
Effects of Fe2+ on the anaerobic digestion of chicken manure: a batch study
.
Third International Conference on Digital Manufacturing & Automation
doi: 10.1109/ICDMA.2012.259
.

Supplementary data