Short communication
Analysis of the microbiota associated with the blackening of activated sludge

https://doi.org/10.1016/j.ibiod.2020.105140Get rights and content

Highlights

  • Diverse microorganisms previous unknown are responsible for sludge blackening.

  • More indigenous microbiota of the activated sludge is implicated to producing sulfide.

  • Diverse microbes are key players involving ecological S–N–C transformation.

Abstract

Activated sludge blackening is an alarming issue encountered during the operation of biological treatment processes, but the microbiota associated with the blackening of activated sludge remains unknown. 16S rRNA gene amplicon sequencing was applied to reveal the microbial community composition and dynamics of activated sludge experiencing the sludge blackening in this study. Surprisingly, microorganisms proliferated significantly in blackened sludge are not known to sulfur metabolism based on the information available. Functional profiles indicated that microbial communities of all 14 sludge samples have potential to reduce sulfate to sulfide. Genome annotation further validated microorganisms affiliated with Acidovorax and Mycolicibacterium were responsible for sulfide production in all samples. The homologues of Pseudomonas fluorescens, Janthinobacterium lividum, Hydrogenophaga intermedia, and Mycolicibacterium pulveris were potential active sulfide producer dominated the blackened sludge. This study revealed a more diverse sulfide producing microbial community unknown previously and further delineation of their metabolic versatilities including denitrification, sulfate reduction to sulfide, and thiosulfate oxidation to sulfate is necessary. Findings from this study suggested a broad role and contribution of these generalist microorganisms in regulating ecological sulfur-nitrogen-carbon transformation. Knowledge from such research offer in-depth understanding of the biochemical reaction responsible for sludge blackening and inspired novel research focuses on microbial-driven sulfur-nitrogen-carbon cycle.

Introduction

The blackening of activated sludge is a common issue encountered during the operation of biological treatment processes, which in general accompanied with process deterioration. Sludge blackening takes place in a pilot activated sludge plant due to a sudden change of the influent sewage composition (Hardy et al., 2001). As a result, the process showed declined removals of ammonia and chemical oxygen demand (COD), and high hydrogen sulfide was detected concurrently. Sludge turned black in a simultaneous anammox and denitrification process due to unguarded temporary mixing malfunction (Takekawa et al., 2014). Shi et al. (2017) reported the anammox sludge turned from red to black color within three weeks when the reactor was loaded with 2 mg/L oxytetracycline, and deprived nitrogen removal performance was obtained consequently. In addition, the blackening of water and sludge associated with the rotten-egg smell has been also reported in many inland rivers and lakes (Li et al., 2019; Song et al., 2017). Existing studies are limited to phenomenon report regarding the sludge blackening. Investigations relevant to this omnipresent incident have been overlooked.

Currently, the formation of hydrogen sulfide is being deemed as the cause of the sludge blackening and process deterioration given that the hydrogen sulfide inhibits or inactivates microorganisms (Reiffenstein et al., 1992). The sulfate reducing bacteria (SRB) have been generally accused as the evildoer leading to the blackening of activated sludge cause the SRB is well known for its sulfide production capability (Muyzer and Stams, 2008; Papi et al., 2018). However, this widely circulated creed is challenged by the horse sense that the classical SRB (e.g., Desulfococcus, Desulfobulbus, Desulfovibrio, and Desulfobacter) (Muyzer and Stams, 2008) are neither the leading microbe in conventional nitrification/denitrification processes (Hardy et al., 2001) nor in innovatory wastewater treatment processes such as partial nitritation/anammox (Shi et al., 2017; Takekawa et al., 2014; Wang et al., 2018). Thus, there is high possibility that microorganisms in disguise rather than conventional SRB take the responsibility for hydrogen sulfide generation. Besides, burying in mind, the aforesaid studies have not investigated the microbial community dynamics during the sludge blackening, and microbes responding to hydrogen sulfide production were unheeded to the ground. Exploiting the microbial community associated with the blackening of activated sludge offers insights into the disguised sulfide producing microorganisms and provides technical basis for process control. Such aspects demand extensive studies.

In this study, blackened sludge samples were randomly collected from the laboratory, and the microbial community variation during sludge blackening was scrutinized. Functional profiles prediction, genome annotation, and metabolic pathway reconstruction were applied to exploit the microbiota associated with the sludge blackening. The core objectives of this study consist 1) identifying functional microorganisms responding to hydrogen sulfide production; and 2) inspecting the microbial community dynamics during the sludge blackening.

Section snippets

Sample collections

In this study, 14 biomass samples from four provenances were collected for microbial community determination (Table 1). The blackened sludge samples (i.e., P1–P7) of the first three groups are in-situ precipitates in individual 20-L buckets filling with three types of wastewater. The relevant wastewater sources are effluent sewage of the preliminary treatment process (i.e., screening and degritting) with FeCl3-enhanced coagulation sedimentation treatment (Group 1), effluent from the secondary

Microbial community dynamics

The microbial community diversity determined significant evolutions along with sludge blackening. Table 2 summarized that Good's coverage >91.5% was obtained for all samples, manifesting a satisfactory sequencing depth. The alpha diversity analysis indicated both richness and diversity indices remarkably reduced whilst the sludge turned black as for Group 1 and Group 3. As for samples from Group 2, the richness showed a decreasing trend, however, the diversity indices fluctuated in between

Conclusions

Diverse microorganisms in disguise rather than conventional SRB have been identified as the sulfide producer leading to the sludge blackening. Indigenous bacteria of activated sludge, i.e., Acidovorax sp. and Mycolicibacterium sp., were revealed to be capable of producing sulfide. Most of detected sulfide producing microorganisms in blackened sludge are competent to perform sulfate reduction to sulfide and denitrification. Ubiquitous bacteria such as Pseudomonas fluorescens, Acidovorax caeni,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was financially supported by Guangdong Provincial Key Laboratory of Fishery Ecology and Environment (FEEL-2020-11), Guangxi Young Research Ability Improvement Project (2019KY0242), and National Natural Science Foundation of China (21806110).

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