Characterizing the growing microorganisms at species level in 46 anaerobic digesters at Danish wastewater treatment plants: A six-year survey on microbial community structure and key drivers.

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting primary and surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive datasets have hindered a deeper understanding of the characteristics and associations between key variables and the microbial community composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the first and largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. High-resolution bacterial and archaeal community analyses were carried out at species level using amplicon sequencing of >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera, and 3,584 species in the bacterial community, where 70% of the genera and 93% of the species represented environmental taxa that were only classified based on MiDAS 3 de novo placeholder taxonomy. More than 40% of the bacterial species were found not to grow in the mesophilic and thermophilic digesters and were only present due to immigration with the feed sludge. Ammonium concentration was the main driver shaping the bacterial community while temperature and pH were main drivers for the archaea in the three types of ADs. Sub-setting for the growing microbes improved significantly the correlation analyses and revealed the main drivers for the presence of specific species. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with specific growing species. This survey provides a comprehensive insight into community structure at species level, providing a foundation for future studies of the ecological significance/characteristics and function of the many novel or poorly described taxa.

Identification of microorganisms responsible for foam formation in mesophilic anaerobic digesters treating surplus activated sludge.

Foaming is a common operational problem in anaerobic digestion (AD) systems, where hydrophobic filamentous microorganisms are usually considered to be the major cause. However, little is known about the identity of foam-stabilising microorganisms in AD systems, and control measures are lacking. This study identified putative foam forming microorganisms in 13 full-scale mesophilic digesters located at 11 wastewater treatment plants in Denmark, using 16S rRNA gene amplicon sequencing with species-level resolution and fluorescence in situ hybridization (FISH) for visualization. A foaming potential aeration test was applied to classify the digester sludges according to their foaming propensity. A high foaming potential for sludges was linked to the abundance of species from the genus Candidatus Microthrix, immigrating with the feed stream (surplus activated sludge), but also to several novel phylotypes potentially growing in the digester. These species were classified to the genera Ca. Brevefilum (Ca. B. fermentans) and Tetrasphaera (midas_s_5), the families ST-12K33 (midas_s_22), and Rikenellaceae (midas_s_141), and the archaeal genus Methanospirillum (midas_s_2576). Application of FISH showed that these potential foam-forming organisms all had a filamentous morphology. Additionally, it was shown that concentrations of ammonium and total nitrogen correlated strongly to the presence of foam-formers. This study provided new insight into the identity of putative foam-forming microorganisms in mesophilic AD systems, allowing for the subsequent surveillance of their abundances and studies of their ecology. Such information will importantly inform the development of control measures for these problematic microorganisms.

MiDAS 3: An ecosystem-specific reference database, taxonomy and knowledge platform for activated sludge and anaerobic digesters reveals species-level microbiome composition of activated sludge.

The function of the microbiomes in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of their members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain), high resolution taxonomic classification is crucial to understand the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on full-length 16S rRNA gene amplicon sequence variants (FL-ASVs) derived from activated sludge and anaerobic digester systems in Denmark. The new database proposes unique provisional names for all unclassified microorganisms down to species level, providing a new and much-needed tool for microbiome research. The MiDAS 3 database was used to analyze the microbiome in 20 Danish wastewater treatment plants with nutrient removal, sampled over 13 years. The 50 most abundant species belonged to 42 genera, including 14 genera with provisional 'midas' name. Of those, 20 have no known function in the system, which highlights the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The new MiDAS 3 database also forms the backbone of the MiDAS Field Guide - an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (>1800) and species (>4200) found in activated sludge and anaerobic digesters in Denmark, but is also relevant to wastewater systems across the world. The identity of the microbes is linked to functional information, where available, and the website provides the possibility to BLAST new sequences against the MiDAS 3 database. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository, facilitating understanding of wastewater treatment ecosystem function.

Candidatus Amarolinea and Candidatus Microthrix Are Mainly Responsible for Filamentous Bulking in Danish Municipal Wastewater Treatment Plants.

Filamentous bulking is a common serious operational problem leading to deteriorated sludge settling that has long been observed in activated sludge biological wastewater treatment systems. A number of bacterial genera found therein possess filamentous morphology, where some have been shown to be implicated in bulking episodes (e.g., Ca. Microthrix), the impact of many others is still not clear. In this study we performed a survey of 17 Danish municipal wastewater treatment plants (WWTPs) with nutrient removal using 16S rRNA amplicon sequencing over a period of 13 years, where all known filamentous bacteria from 30 genera were analyzed. The filamentous community constituted on average 13 ± 6%, and up to 43% of total read abundance with the same genera common to all plants. Ca. Microthrix and several genera belonging to phylum Chloroflexi were among the most abundant filamentous bacteria. The effect of filamentous bacteria on sludge settling properties was analyzed using measurements of the diluted sludge volume index (DSVI). Strong positive correlations with DSVI were observed only for Ca. Microthrix and Ca. Amarolinea, the latter being a novel, recently characterized genus belonging to the phylum Chloroflexi. The bulking potential of other filamentous bacteria was not significant despite their presence in many plants. Low phylogenetic diversity was observed for both Ca. Microthrix and Ca. Amarolinea, making physiological characterization of individual species and potential development of control strategies more feasible. In this study we show that, despite the high diversity of filamentous phylotypes in Danish WWTPs, only few of them were responsible for severe bulking episodes.

Monitoring foaming potential in anaerobic digesters.

Foaming in anaerobic digestion (AD) systems for biogas generation can give serious operational problems. The cause of such foaming events is often unclear, and it is therefore not an easy task to predict and subsequently apply preventative measures. Methods for the measurement of the foaming potential of digester sludge are often implemented, but no standardized method is available. In this study, we investigated parameters influencing the foam formation during experimental aeration tests of full-scale digester sludge, including air flow, time, and total solids concentration, and proposed an optimized method for standard use. In a survey of 16 full-scale AD systems located at wastewater treatment plants in Denmark, all sludge samples were classified into three groups (non-foaming, pre-foaming, and actually foaming) according to their foam height/propensity and stability. Extensive surveillance of plants with the proposed classification system will enable the determination of cut-off values to help to identify foaming or pre-foaming sludge, and to associate these with operational conditions leading to foaming episodes.

Can Chlorella pyrenoidosa be a bioindicator for hazardous solid waste detoxification?

Four kinds of solid waste residue (SWR, S1 to S4) from different stages in a sequential detoxification process were chosen. The biotoxicity of the leachates from S1 to S4 was tested by Chlorella pyrenoidosa. The growth inhibition, the chlorophyll a (chla) and chlorophyll b (chlb) concentrations, and the ultrastructural morphology of cells of C. pyrenoidosa were studied. It shows that the growth inhibition of C. pyrenoidosa significantly increased with increasing leachate concentration when exposed to the leachates from S1, S2, S3, and S4, respectively. It well reflects the toxicity difference of leachate from SWR at different treatment stages, namely S1>S2>S3>S4. Correspondingly, the chla and chlb concentrations of C. pyrenoidosa increased gradually as SWR was treated deeply. Leachate disrupted chlorophyll synthesis and inhibited cell growth. The changing of the ultrastructural morphology of cells under different leachate exposures, such as volume of chloroplasts and quantity of thylakoids reducing, confirmed the toxicity decrease of leachates from different stages. C. pyrenoidosa is a good bioindicator for hazardous solid waste detoxification. The EC(50) at difference scenarios also suggests that it was feasible to estimate ecological toxicity of leachates to C. pyrenoidosa after exposure times of 72h. C. pyrenoidosa can be introduced to evaluate the effect of hazardous solid waste disposal by biotoxicity assessment.

Leaching behavior of iron from simulated landfills with different operational modes.

The aim of the present study was to investigate the leaching behavior of iron from simulated landfills with different operation modes, with an emphasis on the variation of iron in different oxidation state, ferrous Fe(II) and ferric Fe(III) percentage and the distribution of iron content in different landfill leachate fractions. The leaching behavior and accumulated amounts of iron leached out by leachate from conventional landfill (CL) and leachate recirculated landfill (RL) exhibited decidedly different trends except for the initial 28 days. In addition, the percentage of iron leached from CL and RL accounted 1.00% and 0.14% for the total amount in landfills, respectively. No correlations between iron and selected characteristics in leachate were found were observed in the two simulated landfills. Significant positive correlations between particulate bound iron and Fe(III) were found in the leachates from RL (R(2)=0.748) and CL (R(2)=0.833).

Releasing behavior of copper in recirculated bioreactor landfill.

The purpose of this study was to determine the releasing behavior of copper in municipal solid waste (MSW) in landfill with respect to refuse and leachate as an inseparable system. Two simulated bioreactor landfills, one with leachate recirculation and the other without, were operated in room temperature for 320 days. Copper in refuse showed behaviors of staggered migration and retention, which corresponded with the degradation process of landfill obviously. The significant different amounts of Cu2+ leached out from refuse into leachate of two landfills were 24.74 mg and 118.53 mg after 320 days' operation, respectively. It also reflected the releasing behavior of copper in landfill refuse at different stage accordingly. The results confirmed that the refuse in landfill had high potential of secondary pollution after closure.

Responses of oxidation rate and microbial communities to methane in simulated landfill cover soil microcosms.

CH4 oxidation capacities and microbial community structures developed in response to the presence of CH4 were investigated in two types of landfill cover soil microcosms, waste soil (fine material in stabilized waste) and clay soil. CH4 emission fluxes were lower in the waste soil cover over the course of the experiment. After exposure to CH4 flow for 120 days, the waste soil developed CH4 oxidation capacity from 0.53 to 11.25-13.48micromol CH4gd.w.(-1)h(-1), which was ten times higher than the clay soil. The topsoils of the two soil covers were observed dried and inhibited CH4 oxidation. The maximum CH4 oxidation rate occurred at the depth of 10-20cm in the waste soil cover (the middle layer), whereas it took place mainly at the depth of 20-30cm in the clay soil cover (the bottom layer). The amounts of the phospholipid fatty acid (PLFA) biomarks 16:1omega8c and 18:1omega8c for type I and II methanotrophs, respectively, showed that type I methanotrophic bacteria predominated in the clay soil, while the type II methanotrophic bacteria were abundant in the waste soil, and the highest population in the middle layer. The results also indicated that a greater active methanotrophic community was developed in the waste soil relative to the clay soil.

[Effects of methane stress on oxidation rates and microbial community structures in different landfill cover soils].

As compared with the ordinary landfill cover material, clay soil, the effect of methane stress on oxidation rate and microbial community structure was investigated in waste soil (material from biologically treated municipal solid waste). The results showed that the moisture content of the clay soil was low, due to the low water retaining capacity. As environmental temperature and rainfall changed, the clay soil caked and inhibited methanotrophs growth. However, with a high organic matter, water-holding capacity and porosity, the waste soil provided a favor condition for methanotrophs growth and propagation. After exposure to methane flow for 120 days, methane oxidation potential in the middle and bottom layers of the waste soil column increased to 11.25-13.48 micromol/(g x h), which was 10.4-24.5 times higher than that in clay soil column. The topsoils were both found to be dried and inhibit methane oxidation. Methane oxidation (removal) efficiency by the waste soil column reached 48.3% at the end of the experiment, which was 5-6 times higher than that by the clay soil column. The amounts of the phospholipid fatty acid (PLFA) biomarks 16:1 omega 8c and 18:1 omega 8c for Type I and II methanotrophs, respectively, showed that a strong linear relationship was observed between methane oxidation potential and PLFA 18:1 omega 8c content in soil samples.