Methane Promotion of Waste Sludge Anaerobic Digestion: Effect of Typical Metal Meshes on Community Evolution and Electron Transfer

Abstract

Anaerobic digestion of waste activated sludge (WAS) to produce methane is a promising pathway for biomass energy recovery. However, a slow organic biodegradation rate and weak microbial cooperation between fermentation bacteria and methanogens lead to low methane production from WAS. Considering the reuse of conductive materials for the regulation of microbial communities, this study chose three kinds of high-mesh metal materials (nickel, copper, and stainless steel) to promote the anaerobic digestion process. All three kinds of metal mesh could effectively increase methane production, and the highest methane production was increased by 61%, reaching 77.52 mL gVSS⁻¹. The poor biocompatibility of the stainless steel mesh was the least effective in promoting methane production compared to the biocompatible copper mesh and nickel mesh. The microbiological analysis found that the metal mesh with good biocompatibility can effectively induce and promote the enrichment of key microorganisms in the process of synergistic methane production, and the direct electron transfer process (DIET) of microorganisms on the metal surface contributes to the further improvement of the methane production efficiency. Therefore, the application of metal conductive materials in sludge anaerobic fermentation is feasible to achieve the retention of syntrophic bacteria and methanogens in the system.

1. Introduction

As a sustainable and promising technology, anaerobic digestion (AD) is widely used in wastewater treatment and solid waste disposal [1,2]. Through the AD process, microorganisms can convert organic wastes such as waste activated sludge (WAS) to biomethane (CH₄). This helps wastewater treatment plants (WWTPs) reduce their carbon footprint if their energy consumption can be compensated for through CH₄ cogeneration [3]. At present, most WWTPs have built sludge anaerobic fermentation facilities, but their practical operation is far from satisfactory due to poor operational stability and low production efficiency [4].

Introducing conductive materials (CMs) into AD has been proven to be an effective way to enhance methane production and improve system stability [5]. The addition of CMs into the anaerobic digester helps to cope with extreme conditions such as digestion deterioration due to temperature changes, over-acidification, and increased H₂ partial pressure [6]. Generally, CMs can thermodynamically and kinetically facilitate AD to form a more efficient synergistic metabolic process and improve the electron capture capacity of methanogens by replacing diffusive electron mediators (such as acetate, formate, and hydrogen), ultimately accelerating the rate of methanogenesis [7,8]. The electrons generated by organic oxidation can be directly transferred to methanogens through CMs, which is called direct interspecies electron transfer (DIET). DIET has always been considered to be the main reason for the promotion of anaerobic methane production by CMs, although it also has other effects, such as the directional enrichment of functional microorganisms, the regulation of pH stability, and adsorption and immobilization of CO₂ by the surface groups of CMs. These reports imply that the mechanism of methanogenesis via CMs in practical reactors is not yet well understood.

It was found that not only typical electrochemically active bacteria (EAB), such as Geobacter and Pseudomonas, can mediate the DIET process of microorganisms but also some syntrophic microorganisms have the ability to transfer electrons to CMs [5]. These syntrophic microorganisms are critical in anaerobic methanogenesis, as they can convert volatile fatty acids (VFAs) produced by acid-producing bacteria into acetate and hydrogen that are required by methanogens. This “synergistic methanogenesis process” established by methanogens and syntrophic microorganisms is also one of the key factors affecting the rate of methane production. However, these syntrophic microorganisms are relatively rare in conventional AD systems. The effect of CMs on the synergistic methanogenesis process is also uncertain.

To date, a variety of particle CMs, including carbon-based materials [4] and metal nanoparticles [9,10,11,12,13], are reported to significantly improve the performance of AD. In practice, however, the application of particle CMs often encounters material loss and recovery problems [14]. Metal mesh, with good electrical conductivity and high mechanical strength, can be easily fixed in the anaerobic reactors and easily separated from AD residues. Enhancing the AD of WAS by introducing low-cost transition metal meshes (such as nickel (Ni), iron (Fe), and copper (Cu)) are operationally and economically feasible.

In this study, three kinds of metal conductive materials (nickel mesh, copper mesh, and steel stainless mesh) were used to strengthen the methane production process of sludge. In order to promote the hydrolysis of the sludge and prevent the influence of metal corrosion, the sludge was pretreated with alkali, so that the whole process was carried out under weak alkaline conditions. The effects of different metal materials on the substrate metabolism and methanogenesis of the sludge anaerobic digestion process were investigated. Possible DIET processes and the growth of syntrophic microorganisms during methanogenesis were explored. The application and future development of using metal mesh as a method to intensify the AD process is proposed.

2. Materials and Methods

2.1. WAS Characteristics and Pretreatment

All WAS used in this study came from the secondary sedimentation tank of a municipal sewage treatment plant in Harbin, Heilongjiang Province. The ratio of volatile suspended solids (VSS) to total suspended solids (TSS) was approximately 0.62. After being retrieved, the WAS was naturally settled in a refrigerator at 4 °C for 24 h and the supernatant was removed by the siphon method. The concentrated sludge was passed through a 200-mesh sieve to remove large particles such as sand and gravel. The physicochemical indexes of the concentrated sludge after treatment were as follows: VSS of 37.5 g/L, total chemical oxygen demand (COD) of 11,880 mg/L, and pH of 6.90. For the convenience of comparison and calculation, the WAS described above was adjusted to 14.0 gVSS/L by diluting with purified water, and then it was pretreated by alkali to promote the release of intracellular substances. The specific operation was as follows: the original pH of the WAS was adjusted to 10 with 6 mol/L NaOH solution, stirring for 10 min, adjusting the pH of the WAS to 10 again, and repeating the above operation three times [15].

2.2. Reactor Construction and Operation

Eight lab-scale anaerobic fermentation devices were constructed using 550 mL glass bottles with 350 mL of pretreated WAS and 200 mL of headspace (Figure 1A). Each device contained two glass tubes, a short tube for gas collection connected by an air bag and a long tube for liquid sample collection. Nickel mesh (Ni), copper mesh (Cu), and stainless-steel mesh (SS) with a size of 6.25 × 6.25 mm (total area of 200 mm², 0.57 m²_surface/m³_WAS) were put into six of the anaerobic devices. Two devices were set as controls (C) without any metal meshes. The size and specific surface area of the three metal meshes were the same, and their hole diameter was 0.075 mm. The metal meshes were first soaked in 6 mol/L HCl for 24 h before use to remove the surface oxide film. Before operation, all the devices were aerated with N₂ for 10 min to maintain an anaerobic environment and then placed in a constant temperature water bath shaker at 35 ± 2 °C with a shaking frequency of 105 rpm/min. During operation, the reactor does not discharge sludge. A measure of 4 mL of the sludge samples was taken each time for subsequent analysis and detection.

2.3. Analysis and Calculation Methods

2.3.1. Chemical Analysis Method

The VFAs (acetic acid, propionic acid, butyric acid, and valeric acid) [16,17] and gas components (H₂, CO₂, and CH₄) were determined by gas chromatography (7890 A, Agilent Technologies (China) Co.,Ltd., Shanghai, China) equipped with a flame ionization detector and a thermal conductivity detector as described in a previous study [18]. The TSS and VSS were measured by the constant weight method at 105 °C and 600 °C, respectively. The TCOD and COD were determined by HACH spectrophotometry (HACH (Shanghai) Co.,Ltd., Shanghai, China) with a reagent model of 2038315. The soluble carbohydrates were determined by the phenol-sulfuric acid method. Absorbance values were measured at a wavelength of 490 nm using a UV–Vis spectrophotometer (UV-1800, Shanghai MAPADA Instruments Co.Ltd., Shanghai, China). The standard curve was drawn using glucose as the standard substance. The slope of the carbohydrate standard curve was 78.72, and the R² was 0.99. The soluble proteins were determined by a modified BCA protein kit method (Sangon Biotech, Shanghai, China), and the absorbance value of the sample was measured using a UV–Vis spectrophotometer at a wavelength of 562 nm [19]. The standard curve was drawn with bovine serum albumin as the standard substance. The slope of the protein standard curve was 2573, with an R² of 0.99. All the samples for soluble component measurement were centrifuged at 10,000 rpm and filtered through a 0.45 μm filter.

2.3.2. Next-Generation Sequencing and Analysis

At the end of the experiment, metal mesh was taken out from the reactor (Figure 1B), the biofilm formed on metal mesh and the sludge samples were both collected and analyzed. DNA was extracted using a DNA extraction kit (Omega Bio-Tek D55625, Norcross, GA, USA) [20]. The 16S rRNA structures of bacteria and archaea were sequenced using the specific primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [21]. The microbial diversity index, including the Chao1 value, relative abundance (number of OTUs determined in each sample), principal component analysis (PCoA), hierarchical clustering analysis, and function prediction of PICRUSt2, was calculated by I-Sanger online data processing developed by Majorbio (http://www.majorbio.com/ (accessed on 7 September 2022)). The sampling method of biofilm on the surface of the metal mesh was as follows: First, all the metal meshes were removed with tweezers after the experiment, and the floating sludge on the metal surface was removed gently by washing with sterilized phosphate buffered saline (PBS, 10 mM). Second, the washed metal meshes were fully shaken in a vortex shaker for 5 min and centrifuged at 10,000 rpm for 10 min, and the supernatant was removed to obtain a biofilm on the surface of the metal mesh [22].

3. Results and Discussion

3.1. Methane Production Performance

A sequencing batch anaerobic fermentation experiment was used to compare the effects of adding different metal meshes on an AD methane production process. During the entire operation process, the total methane production (Figure 2A), the total carbon dioxide production (Figure 2B), the daily methane production (Figure 2C), and the methane content (Figure 2D) of the experimental groups with the addition of metal materials were significantly higher than those of the control group, indicating that the addition of metal meshes had an effect on methane production in the sludge fermentation process. The experimental group with Ni mesh and Cu mesh obtained the highest cumulative methane production, reaching 380 ± 18 mL (equivalent to 77.52 mL gVSS⁻¹) and 374 ± 16 mL (76.39 mL gVSS⁻¹), respectively, which increased by 61% and 59% compared with the control group. The SS mesh had a less significant effect on methane production, which was only 24% higher than that of the control group. This may be due to the poor biocompatibility of the SS mesh, which was not helpful for microbes attached to participate in DIET or the synergistic methanogenesis process. After several cycles of experiments, the surface of the SS mesh was still bright, and basically no microbial growth was found on it. Furthermore, the cumulative carbon dioxide production in the SS, Ni, and Cu groups was significantly higher than that of the control group, indicating that the addition of metal meshes had a certain promotion effect on the decomposition and mineralization of complex organic matter.

Methane production rate and biogas methane concentration of the experimental group with added metal mesh were significantly higher than those of the control group. With the growth and proliferation of methanogens, the daily methane production increased gradually and reached maximum level during day 6–11. After that, due to the consumption of volatile fatty acids, the daily methane production decreased. The maximum daily methane production of the experimental group with Cu mesh reached 0.123 m³/(m³·d), which was 37% higher than that of the control group. The average methane content of the added metal mesh reached 54%, which was 16% higher than that of the control group. In previous studies, the nanoparticles of three metals were added to the AD system of WAS, and different promoting effects were obtained. For instance, Su et al. [23] obtained a CH₄ increase by >40% by adding 20 nm Fe at a high concentration (1000 mg/L). Panagiotis et al. [24] added Ni-nanoparticles into WAS and slightly (~8%) enhanced the methane yield. Tareq et al. [13] reported a maximum three times increase in methane production by adding 1500 mg/L copper–iron bimetallic nanoparticles. There were also some studies reporting the negative effect of metal nanoparticles on AD [11,25]. Compared with metal nanoparticles, the low-cost metal meshes can also effectively promote methane production from WAS, and according to the results in this study, the promotion effect was Ni mesh ≈ Cu mesh > SS mesh.

3.2. Soluble Organic Matter Variation

The main dissolved organic matter in the sludge mainly includes carbohydrates, proteins, and VFAs [26]. It showed that the addition of metal meshes had little effect on the concentrations of carbohydrates and proteins. During the whole fermentation process, the total carbohydrates concentration was maintained at approximately 200 mg/L (Figure 3A), and the protein concentration was maintained between 450 mg/L and 550 mg/L (Figure 3B).

Various VFAs, mainly including acetic acid (Figure 4A), propionic acid (Figure 4B), iso-butyric acid (Figure 4C), n-butyric acid (Figure 4D), iso-valeric acid (Figure 4E), and n-valeric acid (Figure 4F) were produced from the fermentation of carbohydrate and protein. The concentration of VFAs was affected by both the fermentative acid-producing process and the acid-utilizing process. In early stage, the acid production rate was higher than the consumption rate, thus an accumulation of VFAs was observed, while in the later stage, the situation was just the opposite. Among these VFAs component, only acetic acid can be directly utilized for methanogens, hence its consumption was first observed, whose concentration rapidly decreased after Day 2. From Day 4 to Day 6, it showed that the acetic acid concentration in the control group decreased from 747 mg/L to 512 mg/L, while the acetic acid concentration in the Ni mesh decreased from 582 mg/L to 146 mg/L. This suggested that the degradation rate of acetic acid was significantly accelerated by metal mesh addition.

Except for acetic acid, other VFAs need to be converted into acetic acid before they are used for methane production. Among them, the acetogenesis of butyric acid is relatively easy, followed by valeric acid, and propionic acid is thermodynamically most unfavorable. Therefore, other VFAs were degraded in preference to propionic acid. The VFAs concentrations in the Ni and Cu group were lower than those of the control group most of the time, and after approximately 10 days, all organic acids were degraded and converted to methane, suggesting that metal meshes accelerated the conversion rates compared to the control groups. Little residual propionic acid was detected, and its concentration in the Cu and Ni mesh groups was 20–80 mg/L, while it was 121 mg/L in the control group. These results showed that the addition of Ni mesh and Cu mesh could accelerate the synergistic methanogenesis process in sludge, which was consistent with the change in methane production rate. When acetic acid and butyric acid were exhausted (at day 8), the Ni and Cu groups still maintained a high methane production rate. This proved that the SS mesh showed a weak promotion effect on VFAs conversion compared to the Ni and Cu meshes. This may be caused by the poor biocompatibility of SS, as biomass retention is crucial for the synergistic metabolic process of microorganisms [27].

3.3. Effects of Metal Meshes on the Microbial Community

In PCoA analysis, the relative distance of biofilms formed on the surfaces of Ni mesh (Ni-biofilm) and SS mesh (SS-biofilm) differed from their sludge community structure (Figure 5A). Therefore, a large surface of meshes would extend the promotion effect in the system compared to granular carriers. Furthermore, the relative abundance of some functional bacteria in sludge changed significantly by metal mesh additions. As shown in Figure 5B, with metal mesh additions, the relative abundance of Clostridiales decreased significantly, while Anaerolineales and Synergistales increased significantly, both of which were proven to be key syntrophic microorganisms involved in methanogenesis during AD. The relative abundance of Anaerolineales increased from 1.79% to 8.27% in Ni-sludge. Many genera in Anaerolineales have been confirmed to utilize a variety of substrates, such as sugars, peptides, and organic acids [28], and Synergistales have been confirmed to interact with hydrogenotrophic methanogens for methane production [29]. Hence, their existence was beneficial to promote the synergistic methanogenesis process.

It was Interesting to show that SS mesh had a great influence on sludge communities, while Ni-sludge, Cu-sludge, and Control-sludge were quite similar. Analysis of archaeal community structure found that highly enriched communities of Methanobacteriales (0.91–0.96%) and Methanosarcinales (2.53–3.33%) were found in Ni-sludge and Cu-sludge compared to the Control-sludge (0.35–0.75%), while no significant enrichment of methanogens was found in SS-sludge. Some different enrichment of several fermentative microorganisms was observed in SS sludge, including Thermomicrobiales, Caldilineales, Synergitales, and Blastocatellales, rather than in the sludge with Ni or Cu mesh addition.

The clustering analysis of genera showed that the addition of metal materials played a positive role in the enrichment of some fermentative bacteria and methanogens in the sludge (Figure 6). The addition of Ni mesh and Cu mesh significantly increased the abundance of norank_f_Anaerolineaceae [30] (1.95–4.29%) and Longilinea (1.43–2.42%) compared to the control (0.50–0.97%). Longilinea has been shown to release intracellular electrons through the metabolism of substrates or intermediates [31,32]. The fermentive bacteria Unclassified_f_Blastocatellaceae (1.50%) [33] and Lactivibrio (1.71%) were more dominant in SS sludge than others (<0.14%). The ecological function of Lactivibrio has been confirmed to participate in the metabolism of carbohydrates and VFAs to provide substrates for methanogens [34]. Therefore, the enrichment of these genera can provide abundant substrates for methanogens, thereby promoting methanogenesis. The methanogens detected in the sludge mainly included the following five species: Methanosaeta, Methanosarcina, Methanobacterium, Methanobrevibacter, and Methanospirillum. The most dominant Methanosaeta, as a kind of acetotrophic methanogen, was significantly enriched by metal mesh additions, which was increased from 0.63% to 1.96% and 2.82% in Ni-sludge and Cu-sludge compared to the control. These results suggested that the addition of metal mesh could promote the growth of syntrophic microorganisms and Methanosaeta, which may be responsible for the promotion of methanogenesis.

3.4. Analysis of the Potential DIET and Synergistic Methanogenesis Process Mediated by Metal Mesh

The metal mesh surface finally formed a different community structure from the sludge community, which meant that there might be different methanogenesis pathways on the metal surface. The top 20 families with significant differences between the metal surface and the corresponding sludge community in Ni mesh group (Figure 7A) and Cu mesh group (Figure 7B) were listed. A relatively higher level of Bacteroidetes_vadinHA17, Methanosaetaceae and Methanobacteraceae was found on both Ni and Cu surfaces compared to the corresponding sludge. Bacteroidetes_vadinHA17 is known as an electrogenic class for direct electricity production, and Methanosaetaceae and Methanobacteraceae were confirmed to be involved in the DIET process of methanogenesis, both of which have been extensively detected in other CM-enhanced systems [35]. Correspondingly, typical fermentative bacteria on the metal surfaces, such as Peptrostreptococcaceae and Clostridiaceae_1, were decreased significantly, while the proportion of some typical methanogenic syntrophic microorganisms, such as Anaerolineaceae, Spirochaetaceae, and Syntrophomonadaceae on the Cu-surface and Rhodocyclaceae on the Ni-surface, increased. Rhodocyclaceae were potentially electrically syntrophic with Methanosaeta via DIET and were selectively enriched in a magnetite-mediated AD system [36]. Hence, it was possible that the high electrical conductivity of metals selectively enriched these genera. The major genera identified were Longilinea (1.93% and 1.41%), Syntrophomonas (0.85% and 0.72%), and Smithella (0.70% and 0.67%), which were identified as potentially involved in the DIET process [32,37,38].

To accurately predict the variation in functional modules of the methanogenesis process associated with DIET, COG functional classification was analyzed using the PICRUSt2 model based on 16S rRNA gene sequencing of sludge and metal surface communities (Figure 8). The results showed that the addition of metal meshes enhanced metabolic activities, including translation, ribosomal structure and biogenesis (J), posttranslational modification, protein turnover, chaperones (O) and intracellular trafficking, secretion, and vesicular transport (U). The activity of Ni-surface and Cu-surface was the highest, followed by the corresponding sludge group and control group. Furthermore, functions responsible for hydrogenotrophic and electron transfer activities, cell wall/membrane/envelope biogenesis (M) and coenzyme transport and metabolism (H) were enhanced, suggesting that the synergistic metabolism and DIET processes were improved. These results showed that the surface of metal materials could promote the growth of methanogens and other syntrophic microorganisms with extracellular electron transfer ability, which was conducive to the formation of DIET and the synergistic methanogenesis process between fermentative microorganisms and methanogens.

3.5. Mechanism Analysis of Metal Mesh Promoting Methane Production

The results of this study demonstrated that using metal meshes could promote the growth of methanogens and syntrophic bacteria not only on the surface but also in sludge communities. The relative abundance of the functional microorganisms for methanogenesis on the surface of the metal mesh was higher than that in the corresponding sludge. This result indicated that the SS mesh with poor biocompatibility for methanogens and syntrophic bacteria was significantly lower than that of the Ni mesh and Cu mesh with good biocompatibility, and a much lower methane yield was obtained by SS. The possible microorganisms that participated in the DIET process on the metal surface are shown in Table 1. The relative abundance of Methanosaetaceae, which could directly participate in the DIET process, increased by 6–7 times on the metal surface, and the relative abundance of other methanogens, such as Methanosaetaceae, Methanobacteriaceae, and Methanospirillaceae, all increased. Syntrophomonadaceae and Syntrophaceae are key bacteria in syntrophic methanogenesis and contain many genera that can metabolize long-chain fatty acids (LCFAs) into acetate and hydrogen. They were significantly enriched on the surface of the material (1.27–1.43% and 0.90–0.93%) but were rarely present in the sludge (0.41–0.74% and <0.04%). Spirochaetaceae are apparently involved in the synergistic acetic acid oxidative (SAO) methanogenesis process of hydrogenotrophic methanogens. Pseudomonadaceae had the ability of extracellular electron transport and was enriched on metal surfaces with a relative abundance of ~1% but was not found in sludge. These microorganisms are the dominant species for other dosing of CMs, such as magnetite and granular activated carbon, to induce the DIET process [39,40]. These microorganisms can utilize the high conductivity of CMs to complete the DIET methanogenesis process and induce the sludge to form a better synergistic methanogenesis process, thereby significantly increasing the methanogenesis rate (Figure 9). The enrichment of methanogens and related syntrophic microorganisms observed in this study through the addition of metal meshes is an important reason for improving the methane production rate of sludge, which shows its potential for accelerating the methane production of sludge. The metal mesh can be applied to the AD system in the form of suspended filler, which can be easily recovered and reused by means of sieve filtration. In this way, it is expected to realize the retention of methanogens and syntrophic bacteria in the system and finally realize the rapid production of methane.

4. Conclusions

The results of the study showed that the application of metal mesh could significantly promote the methanogenesis process of sludge. The methane yield of sludge with the addition of Ni mesh and Cu mesh increased the methane production by 61% and 59%, respectively. Community and metabolic analysis found that the introduction of metal meshes significantly enhanced the metabolic process of VFAs by promoting the growth of the syntrophic microorganisms. The microorganisms involved in the DIET and synergistic methanogenesis process were enriched, including Bacteroidetes_vadinHA17, Anaerolineaceae, Synergistaceae, Rhodocyclaceae, and Methanosaetaceae. Comparing the effects of different metal meshes on AD, it was found that biocompatibility was more important than electrical conductivity. The engineering implication of metal meshes in anaerobic fermentation is that they can be recycled and reused. This was a preliminary discussion, and more specific optimization experiments should be carried out continuously to obtain more valuable results.

Author Contributions

Writing and original draft preparation, L.W.; software, C.L.; methodology, X.F.; review and editing, C.Y.; validation, X.Z.; conceptualization, Z.G. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

WAS	Waste activated sludge	DIET	Direct electron transfer process
AD	Anaerobic digestion	WWTPs	Wastewater treatment plants
CMs	Conductive materials	EAB	Electrochemically active bacteria
VFAs	Volatile fatty acids	VSS	Volatile suspended solids
TSS	Total suspended solids	COD	Chemical oxygen demand
SS	Stainless steel	PCoA	Principal component analysis
PBS	Phosphate buffered saline	LCFAs	Long-chain fatty acids
SAO	Synergistic acetic acid oxidative

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Figures and Table

Figure 1. Schematic diagram of fermentation devices (A) and metal meshes after experiment (B).

Figure 2. Total methane production (A), carbon dioxide production (B), daily methane production (C), and methane content (D) with different metal meshes.

Figure 3. Changes of total (A) soluble carbohydrates and (B) soluble proteins in sludge.

Figure 4. The changes of (A) acetic acid, (B) propionic acid, (C) iso-butyric acid, (D) n-butyric acid, (E) iso-valeric acid, and (F) n-valeric acid concentration during AD reactor operation with different metal meshes.

Figure 5. Principal coordinates analysis (PCoA) based on OTUs (A) and the differences in relative abundance of sludge microbial community at order level (B).

Figure 6. Heatmap of sludge community structure at genus level.

Figure 7. Community differences between (A) Ni surface or (B) Cu surface and corresponding sludge.

Figure 8. COG function classification based on the 16S rRNA gene sequencing of sludge and metal surface communities.

Figure 9. Possible methanogenic process in metal meshes mediated AD (DIET: direct interspecies electron transfer, EAB: electrochemical active bacteria, AM: acetotrophic methanogens, HM: hydrogenotrophic methanogens, Syn-M, syntrophic microorganisms).

Table 1

Possible microorganisms that participate in the DIET process on metal surface.

Family	Relative Abundance (%)
	Control	SS-Sludge	Ni-Sludge	Cu-Sludge	Ni-Surface	Cu-Surface
Methanosaetaceae	0.63	0.14	1.96	2.82	4.53	4.29
Methanobacteriaceae	0.35	0.43	0.91	0.96	1.41	1.37
Methanosarcinaceae	0.12	0.05	0.57	0.51	0.17	0.21
Methanospirillaceae	0.06	0.02	0.05	0.21	0.70	0.82
Syntrophomonadaceae	0.74	0.41	0.48	0.58	1.27	1.43
Syntrophaceae	0.04	0.01	0.04	0.03	0.90	0.93
Pseudomonadaceae	0.04	0.02	0.10	0.03	1.01	0.74
Anaerolineaceae	1.79	1.60	8.27	4.39	8.18	6.28
Synergistaceae	0.53	3.78	1.31	1.74	1.13	1.29
Rhodocyclaceae	0.30	0.42	0.27	0.45	2.64	0.72
Spirochaetaceae	0.23	0.09	0.20	0.32	1.43	1.85

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