Minimizing extracellular DNA improves the precision of microbial community dynamic analysis in response to thermal hydrolysis

https://doi.org/10.1016/j.biortech.2020.122938Get rights and content

Highlights

Abstract

Extracellular DNA (exDNA) can induce bias when evaluating the microbiota in wastewater treatment systems, particularly when cell lysis caused by thermal hydrolysis pretreatment (THP) releasing abundant DNA. However, the influence of such exDNA is still unknown. Accordingly, this study applied a pretreatment strategy for DNA extraction with proteinase K and DNase Ⅰ to minimize the influence of exDNA when evaluating the sludge microbiota. Lactobacillus and Peptostreptococcus were confirmed as the main THP-resistant microorganisms. Gram-positive bacteria were more resistant to THP, implying that the presence of a cell wall could promote THP resistance in bacteria. Moreover, the ability to form spores did not affect the resistance of bacteria to THP. These findings showed that resistant microbiota could be effectively evaluated by excluding exDNA, which can provide important insights into the understanding of microbiota dynamic and the effects of pretreatment on the precision of microbiota analysis in sludge.

Introduction

Anaerobic digestion is an appropriate and effective technique to recover energy from waste-activated sludge (WAS) at wastewater treatment plants. The classical anaerobic digestion process includes three stages: hydrolysis, acidification, and methanogenesis (Zhang et al., 2019a). Microorganisms are generally the main components of WAS and a fair amount of energy input is required to lyse the cells and release organic components (Gavala et al., 2003). Therefore, many pretreatment technologies, including mechanical, thermal, chemical, and biological pretreatments or combinations of these pretreatments, have been developed to disrupt the extracellular polymeric substance matrix and cell wall in order to enhance the disintegration and solubilization of WAS (Zhen et al., 2017).

Thermal hydrolysis pretreatment (THP) is the most widely used commercially available technology for WAS degradation and shows excellent performance in improving the biodegradability of WAS (Barber, 2016). THP enhances the anaerobic digestion of WAS by high-pressure boiling of sludge, followed by rapid decompression (Barber et al., 2012), leading to the solubilization and hydrolysis of macromolecular organic components. The fate and transformation of key dissolved organic matter produced by THP have already been reported (Lu et al., 2018). Few studies have focused on the microbial communities present during the THP sludge anaerobic digestion process (Chen et al., 2018) or the reactor stabilization period (Choi et al., 2018, Zhang et al., 2019a); also, the microbial community profiles of THP sludge remain unclear. Gram-positive bacteria, hyperthermophiles, and certain species of bacteria are extremely resistant to heat owing to their thick cell walls, expression of heat-stable proteins, and protection of endospores (Madigan et al., 2014), which may not be destroyed by THP.

During cell lysis, DNA from disrupted microorganisms is released along with the dissolved organic matter. Conventional DNA extraction protocols cannot distinguish exDNA from intracellular DNA (iDNA). The persistence and integrity of exDNA have been verified in previous studies (Ascher et al., 2009, Carini et al., 2017). Direct DNA extraction from THP sludge for polymerase chain reaction (PCR) and high-throughput sequencing can cause a biased estimation of the microbial composition and diversity owing to large amounts of exDNA. Accurate identification of the microbial communities present in a substrate would provide new insights into the diversity and assembly of the microbiota during anaerobic digestion. Although propidium monoazide was previously used to determine viable microorganisms in activated sludge (Guo & Zhang, 2014), this method was associated with major drawbacks, including a significant difference in the relative proportions in heat-exposed samples (Nocker et al., 2010) and the low efficiency of the method in samples with high turbidity (Fittipaldi et al., 2012). Therefore, heat-treated and highly turbid THP samples require alternative methods to improve the precision of microbial community analysis.

Accordingly, in this study, a method using proteinase K and DNase Ⅰ pretreatment to minimize exDNA in THP sludge was developed. This method was then used to investigate the microbial community structures of THP sludge in response to thermal hydrolysis in order to elucidate the general characteristics of THP-resistant taxa. The method could be applied to ultrasonic-, alkali-, and ozone-pretreated sludges and thus provided insights into the diversity and assembly of the microbiota in the substrate for subsequent anaerobic digestion.

Section snippets

Sample collection

All samples in this study were obtained from a municipal wastewater treatment plant in Beijing, China. Raw sludge samples were collected from the influent of a full-scale THP reactor that had been running for >2 years at the following operational conditions: 160 °C, 6 bar, and 30 min. THP sludge samples were collected from the effluent. Samples were stored at 4 °C before use and treated in the laboratory within 24 h.

Minimizing exDNA in THP sludge

The bacterial genome is a nucleoid with dynamic structural properties and is

Changes in nucleic acids in the supernatant following treatment to minimize exDNA

To investigate the efficiency of exDNA removal, the concentrations of nucleic acids were measured after each step (Fig. 1). The concentration of nucleic acids in THP sludge supernatant was initially 1951 ± 33 ng/μL, which was much higher than that in natural aquatic environments (0.2–88 ng/μL) (Vlassov et al., 2007) and deep-sea sediments (0.31 g DNA/m2 in the top centimeter) (Dell'Anno & Danovaro, 2005). The concentration then decreased rapidly after six washes and reached the target range.

Conclusion

The exDNA released by THP sludge obscured microbial community analysis. A method to minimize exDNA was established and applied for detecting THP-resistant microbiota, i.e., Lactobacillus and Peptostreptococcus, which provided a way to recognize resistant bacteria in WAS after sludge pretreatment and suggested that a properly designed pretreatment could lyse specific resistant bacteria, thereby improving the release of organic components. More generally, the method could be used for DNA

CRediT authorship contribution statement

Lijun Yang: Investigation, Formal analysis, Visualization, Writing - original draft. Wei Li: Investigation, Validation, Resources. Weiwei Cai: Conceptualization, Writing - review & editing. Wei Xing: Writing - review & editing. Fangxu Jia: Writing - review & editing. Hong Yao: Funding acquisition, Project administration, Supervision.

Acknowledgement

This work was supported by Beijing Outstanding Young Scientist Project (C19H100010).

Notes

The authors declare no competing financial interest.

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