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In this study, we successfully demonstrated that 454 pyrosequencing was a powerful approach for investigating the bacterial communities in the activated sludge, digestion sludge, influent, and effluent samples of a full scale wastewater treatment plant treating saline sewage. For each sample, 18,808 effective sequences were selected and utilized to do the bacterial diversity and abundance analysis. In total, 2,455, 794, 1,667, and 1,932 operational taxonomic units were obtained at 3 % distance cutoff in the activated sludge, digestion sludge, influent, and effluent samples, respectively. The corresponding most dominant classes in the four samples are Alphaproteobacteria , Thermotogae , Deltaproteobacteria , and Gammaproteobacteria . About 67 % sequences in the digestion sludge sample were found to be affiliated with the Thermotogales order. Also, these sequences were assigned into a recently proposed genus Kosmotoga by the Ribosomal Database Project classifier . In the effluent sample, we found high abundance of Mycobacterium and Vibrio , which are genera containing pathogenic bacteria. Moreover, in this study, we proposed a method to differentiate the “gene percentage” and “cell percentage” by using Ribosomal RNA Operon Copy Number Database.

This study applied Illumina high-throughput sequencing to explore the microbial communities and functions in anaerobic digestion sludge (ADS) from two wastewater treatment plants based on a metagenomic view. Taxonomic analysis using SILVA SSU database indicated that Proteobacteria (9.52–13.50 %), Bacteroidetes (7.18 %–10.65 %) and Firmicutes (7.53 %–9.46 %) were the most abundant phyla in the ADS. Differences of microbial communities between the two types of ADS were identified. Genera of Methanosaeta and Methanosarcina were the major methanogens. Functional analysis by SEED subsystems showed that the basic metabolic functions of metagenomes in the four ADS samples had no significant difference among them, but they were different from other microbial communities from activated sludge, human faeces, ocean and soil. Abundances of genes in methanogenesis pathway were also quantified using a methanogenesis genes database extracted from KEGG. Results showed that acetotrophic was the major methanogenic pathway in the anaerobic sludge digestion.

Sludge digestion is critical to control the spread of ARGs from wastewater to soil. Fate of ARGs in three pretreatment-AD processes was investigated. UP was more efficient for ARGs removal than AP and THP in pretreatment-AD process. The total ARGs concentration showed significant correlation with 16S rRNA gene. The bacteria carrying ARGs could be mainly affiliated with Proteobacteria. Sewage sludge in the wastewater treatment plants contains considerable amount of antibiotic resistance genes (ARGs). A few studies have reported that anaerobic digestion (AD) could successfully remove some ARGs from sewage sludge, but information on the fate of ARGs in sludge pretreatment-AD process is still very limited. In this study, three sludge pretreatment methods, including alkaline, thermal hydrolysis and ultrasonic pretreatments, were compared to investigate the distribution and removal of ARGs in the sludge pretreatment-AD process. Results showed that the ARGs removal efficiency of AD itself was approximately 50.77%, and if these three sludge pretreatments were applied, the total ARGs removal efficiency of the whole pretreatment-AD process could be improved up to 52.50%-75.07%. The ultrasonic pretreatment was more efficient than alkaline and thermal hydrolysis pretreatments. Although thermal hydrolysis reduced ARGs obviously, the total ARGs rebounded considerably after inoculation and were only removed slightly in the subsequent AD process. Furthermore, it was found that the total ARGs concentration significantly correlated with the amount of 16S rRNA gene during the pretreatment and AD processes, and the bacteria carrying ARGs could be mainly affiliated with Proteobacteria.

The effectiveness and treatment conditions of FeCl 3 - and AlCl 3 -coagulated municipal sewage sludge from chemically enhanced primary treatment (CEPT) using anaerobic digestion (AD) and the structure of microbial community were investigated. The results based on 297 measurements under different operational conditions demonstrate good average AD performance of CEPT sludge, that is, percent volatile solid reduction of 58 %, specific biogas production (or biogas yield) of 0.92 m 3 /kg volatile solids (VS) destroyed, and methane content of 65.4 %. FeCl 3 dosing, organic loading rate, temperature, and hydraulic retention time all significantly affected AD performance. FeCl 3 dosing greatly improved specific methane production (methane yield) by 38–54 % and significantly reduced hydrogen sulfide (H 2 S) content in biogas (from up to 13,250 to <200 ppm), contributing to higher methane recovery and simplified biogas cleaning for power generation. Metagenomic analysis suggested that anaerobic digesters of both CEPT sludge and combined primary and secondary sludge were dominated by Bacteroidetes , Proteobacteria , Firmicutes , Actinobacteria , Thermotogae , and Chloroflexi . However, Methanomicrobia methanogens were better enriched in the anaerobic digesters of CEPT sludge than in the combined sludge. Further, different sources of CEPT sludge with various chemical properties nurtured shared and unique microbial community composition. Combined, this study supports AD as an efficient technology for CEPT sludge treatment and poses first insights into the microbial community structure.

Among processes that control microbial community assembly, microbial invasion has received little attention until recently, especially in the field of anaerobic digestion. However, knowledge of the principles regulating the taxonomic and functional stability of microbial communities is key to truly develop better predictive models and effective management strategies for the anaerobic digestion process. To date, available studies focus on microbial invasions in digesters feed with activated sludge from municipal wastewater treatment plants. Herein, this review summarizes the importance of invasions for anaerobic digestion management, the ecological theories about microbial invasions, the traits of activated sludge microorganisms entering the digesters, and the resident communities of anaerobic reactors that are relevant for invasions and the current knowledge about the success and impacts of invasions, and discusses the research needs on this topic. The initial data indicate that the impact of invasions is low and only a small percentage of the mostly aerobic microorganisms present in the activated sludge feed are able to become stablished in the anaerobic digesters. However, there are still numerous unknowns about microbial invasions in anaerobic digestion including the influence of anaerobic feedstocks or process perturbances that new approaches on microbial ecology could unveil.Key points• Microbial invasions are key processes to develop better strategies for digesters management.• Knowledge on pathogen invasions can improve anaerobic digestion microbial safety.• To date, the number of successful invasions on anaerobic digesters from activated sludge organisms is low.• Feed organisms detected in digesters are mostly inactive residual populations.• Need to expand the range of invaders and operational scenarios studied.

Background Biogenic sulfuric acid (BSA) corrosion damages sewerage and wastewater treatment facilities but is not well investigated in sludge digesters. Sulfur/sulfide oxidizing bacteria (SOB) oxidize sulfur compounds to sulfuric acid, inducing BSA corrosion. To obtain more information on BSA corrosion in sludge digesters, microbial communities from six different, BSA-damaged, digesters were analyzed using culture dependent methods and subsequent polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). BSA production was determined in laboratory scale systems with mixed and pure cultures, and in-situ with concrete specimens from the digester headspace and sludge zones. Results The SOB Acidithiobacillus thiooxidans , Thiomonas intermedia , and Thiomonas perometabolis were cultivated and compared to PCR-DGGE results, revealing the presence of additional acidophilic and neutrophilic SOB. Sulfate concentrations of 10–87 mmol/L after 6–21 days of incubation (final pH 1.0–2.0) in mixed cultures, and up to 433 mmol/L after 42 days (final pH <1.0) in pure A. thiooxidans cultures showed huge sulfuric acid production potentials. Additionally, elevated sulfate concentrations in the corroded concrete of the digester headspace in contrast to the concrete of the sludge zone indicated biological sulfur/sulfide oxidation. Conclusions The presence of SOB and confirmation of their sulfuric acid production under laboratory conditions reveal that these organisms might contribute to BSA corrosion within sludge digesters. Elevated sulfate concentrations on the corroded concrete wall in the digester headspace (compared to the sludge zone) further indicate biological sulfur/sulfide oxidation in-situ . For the first time, SOB presence and activity is directly relatable to BSA corrosion in sludge digesters.

Eukaryotic communities in aerobic wastewater treatment processes are well characterized, but little is known about them in anaerobic processes. In this study, abundance, diversity and morphology of eukaryotes in anaerobic sludge digesters were investigated by quantitative real-time PCR (qPCR), 18S rRNA gene clone library construction and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Samples were taken from four different anaerobic sludge digesters in Japan. Results of qPCR of rRNA genes revealed that Eukarya accounted from 0.1% to 1.4% of the total number of microbial rRNA gene copy numbers. The phylogenetic affiliations of a total of 251 clones were Fungi, Alveolata, Viridiplantae, Amoebozoa, Rhizaria, Stramenopiles and Metazoa. Eighty-five percent of the clones showed less than 97.0% sequence identity to described eukaryotes, indicating most of the eukaryotes in anaerobic sludge digesters are largely unknown. Clones belonging to the uncultured lineage LKM11 in Cryptomycota of Fungi were most abundant in anaerobic sludge, which accounted for 50% of the total clones. The most dominant OTU in each library belonged to either the LKM11 lineage or the uncultured lineage A31 in Alveolata. Principal coordinate analysis indicated that the eukaryotic and prokaryotic community structures were related. The detection of anaerobic eukaryotes, including the members of the LKM11 and A31 lineages in anaerobic sludge digesters, by CARD-FISH revealed their sizes in the range of 2-8 μm. The diverse and uncultured eukaryotes in the LKM11 and the A31 lineages are common and ecologically relevant members in anaerobic sludge digester.

* ORP value from −278.71 to −379.80 mV showed indiscernible effects on methane yield. * Fe(II) and Fe(III) promoted more degradation of proteins and amino acids than Fe 0. * The highest enrichment of Geobacter was noted in samples added with Fe 0. * Cysteine was accumulated during iron enhanced anaerobic sludge digestion. * Both iron content and valence were important for methane production. This study compared effects of three different valent iron (Fe 0, Fe(II) and Fe(III)) on enhanced anaerobic sludge digestion, focusing on the changes of oxidation reduction potential (ORP), dissolved organic nitrogen (DON), and microbial community. Under the same iron dose in range of 0−160 mg/L after an incubation period of 30 days (d), the maximum methane production rate of sludge samples dosed with respective Fe 0, Fe(II) and Fe(III) at the same concentration showed indiscernible differences at each iron dose, regardless of the different iron valence. Moreover, their behavior in changes of ORP, DON and microbial community was different: (1) the addition of Fe 0 made the ORP of sludge more negative, and the addition of Fe(II) and Fe(III) made the ORP of sludge less negative. However, whether being more or less negative, the changes of ORP may show unobservable effects on methane yield when it ranged from −278.71 to −379.80 mV; (2) the degradation of dissolved organic nitrogen, particularly proteins, was less efficient in sludge samples dosed with Fe 0 compared with those dosed with Fe(II) and Fe(III) after an incubation period of 30 d. At the same dose of 160 mg/L iron, more cysteine was noted in sludge samples dosed with Fe(II) (30.74 mg/L) and Fe(III) (27.92 mg/L) compared with that dosed with Fe 0 (21.75 mg/L); (3) Fe 0 particularly promoted the enrichment of Geobacter, and it was 6 times higher than those in sludge samples dosed with Fe(II) and Fe(III) at the same dose of 160 mg/L iron.

The goal of making nutrient removal wastewater treatment energy self-sufficient or even energy producing has become a worldwide accepted goal of technology development. The latest upgrade of the Vienna Main Wastewater Treatment Plant (VMWWTP) with a design capacity of 4 million (M) population equivalent (PE) will produce about 20% more energy on a yearly basis than needed for operation due to a special process scheme. It consists of primary sedimentation, a special 2-stage activated sludge (AS) process configuration where excess sludge is only withdrawn from the first stage AS plant. Raw sludge is subject to mechanical thickening to ∼8% digested sludge (DS) for digestion at high solids concentration. The reject water after nitritation is used for denitritation in the first stage AS plant. This results in markedly reducing the energy requirement for aeration. The design of this last upgrade for energy optimization of sludge treatment is based on the long-term full-scale data from the existing plant, results of mid-term pilot investigations, sound theoretical mass balance calculations and an adapted dynamic model development. All this is presented in this paper. The full-scale upgrade is under construction and will start operating in 2020.

Sludge disintegration is an environmental and industrial challenge that requires intensive research and technological development. Sludge has a complex structure with a high yield of various chemical and biological compounds. Anaerobic digestion is the most commonly used process for sludge disintegration to produce biogas, detoxify sludge, and generate biosolids that can be used in agriculture . Biological cell lysis is the rate‐limiting cell lysis. This review discusses the application of sonolysis as a sludge pretreatment for enhanced anaerobic digestion via three combined processes: thermal destruction, hydrochemical shear forces, and radical oxidation. The mechanistic pathways of sono‐pretreatment to enhance biogas, sludge‐enhanced dewatering, activation of filamentous bacteria, oxidation of organic pollutants, release of heavy metals, reduction of bulking and foaming sludge, and boosting ammonia‐oxidizing bacterial activity are discussed in this review. This article also discusses the use of ultrasound in sludge disintegration, highlighting its potential in conjunction with Fenton and cation‐binding agents, and reviews common large‐scale sonoreactors available on the market.. Sludge disintegration is a key challenge in environmental management, requiring advanced techniques for effective treatment. This paper aims to discuss the ultrasonic disintegration as a pretreatment for sludge to enhance anaerobic digestion. Fundamentals of ultrasonic cavitation, involving thermal destruction, hydromechanical shear forces, and radical oxidation, are discussed. The synergy of ultrasound with chemical treatments like Fenton reagents and cation binding agents is highlighted for improved biogas production and sludge dewaterability. The review also explores large‐scale sono‐reactors and future research directions for optimizing ultrasonic sludge disintegration.