Peng Xuya/Li Lei's team WR of Chongqing University: Integrating meta-omics methods to reveal key functional bacteria that affect anaerobic digestion performance under ammonia stress

‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍‍Image Summary Recently, Peng Xuya/Li Lei's team from the School of Environment and Ecology, Chongqing University published a paper entitled "Integrated multi-omics analyses revealing the key microbial phylotypes affecting anaerobic digestion performance under ammonia stress" in Water Research (IF=11.
236), a well-known journal in the environmental field.
research paper
.

The research combined metagenomics and metaproteomics technology to explore the performance of anaerobic digestion reactor and the response of microecology in the process of endogenous ammonia accumulation.
key functional bacteria, and revealed its metabolic repression mechanism
.

The research results not only provide new insights for the identification of the mechanism of ammonia inhibition of destabilizing microorganisms, but also identify targets for the microbial regulation of ammonia inhibition reactors
.

Introduction Adjusting the energy structure is the key to achieving "carbon neutrality"
.

Anaerobic digestion (AD), a technology that can convert organic waste into sustainable energy, is widely used around the world as it contributes to carbon neutrality
.

However, a large number of nitrogen-containing substances such as proteins and nucleic acids in organic wastes will release ammonia nitrogen during the AD process, which makes the AD process prone to ammonia inhibition and instability
.

In view of the fact that AD is a biochemical process mediated by microorganisms, exploring the microbial mechanism of ammonia inhibiting instability will help to strengthen the AD process and solve the instability problem from the source
.

However, due to the extreme sensitivity of acetic acid metabolism to ammonia nitrogen in the destabilization reactor and the limitations of the analytical technology (high-throughput sequencing) used in the past, the current analysis of the mechanism of ammonia inhibition and destabilization in AD process is not perfect
.

In this study, the accumulation of endogenous ammonia was introduced into the anaerobic digestion reactor through the degradation of kitchen waste, so that the reactor experienced a "stable-destabilization-recovery-completely destabilized (gas production stop)" cycle under different ammonia stress levels.
During the whole process, metagenomics and metaproteomics analysis of microbial samples at different stages were carried out to clarify the sensitivity and adaptability of different metabolic links and functional microorganisms to ammonia nitrogen in the AD process, and to identify key functional bacteria that affect the performance of the system.
reveal its metabolic repression mechanism
.

Figure 1: Response of process parameters to ammonia stress
.

Black vertical dashed lines are used to divide different running stages, and black filled points are microbial sampling points
.

 According to the response of physical and chemical indicators in the process of endogenous ammonia accumulation (Fig.
1), the whole reaction process is divided into 5 stages, including: start-up stage, stable stage (S), instability stage I (I1), recovery stage (R) and Instability II (I2)
.

During the start-up and stabilization phases, the methane yield was stable without significant accumulation of volatile fatty acids (VFAs)
.

With the accumulation of ammonia nitrogen to about 3000 mg N/L, in stage I1, VFAs mainly composed of acetic acid accumulated in the reactor, and the methane yield decreased by 58%
.

However, without any recovery measures, after continuous operation, the reactor entered the R stage, the accumulated VFAs were gradually metabolized completely, and the gas production performance also recovered
.

With the further increase of ammonia nitrogen concentration, VFAs mainly composed of propionic acid accumulated in the reactor from 145 d, and the methane yield dropped by 68%, indicating that the reactor entered the I2 stage and became unstable again
.

Figure 2: PCA analysis based on metagenome (a) and metaproteome (b) results and microbial community differentially expressed proteins (DEPs) profiles (c)
.

The dotted arrow is the direction of succession
.

FC is the multiple of difference
.

Each point in c represents an annotated protein, light yellow and yellow indicate significantly up-regulated protein expression (p < 0.
05 and p < 0.
01), and light blue dots and blue indicate significantly down-regulated protein expression (p < 0.
05 and p < 0.
01) , the black dots indicate that the protein expression was not significantly up- or down-regulated
.

The value above the dashed box is the ratio of the number of DEPs to the number of total proteins
.

 Meta-omics analysis of samples at different stages found that as the reaction progressed, the distribution of genes and proteins in the community underwent obvious succession (Figure 2a and Figure 2b), indicating that long-term endogenous ammonia accumulation induced the functional potential and function of the community.
Changes in protein expression
.

And whether compared with the stationary phase or the recovery phase, the number of down-regulated proteins in the I2 stage was higher than that of the up-regulated proteins (Fig.
2c), indicating that the microbial community at this stage may be significantly inhibited and is heading for decline
.

Figure 3.
Distribution of dominant active microorganisms (relative abundance ≥1%)
.

(a) Hydrolytic and acidogenic bacteria responsible for carbohydrate degradation, (b) Protein and amino acid degrading bacteria, (c) Fatty acid degrading bacteria, (d) Mutual propionic acid oxidizing bacteria, (e) Mutual acetic acid oxidizing bacteria, ( f) Methanogens
.

Microorganisms with meshes in ac have both hydrolysis and acid-producing functions, while those without meshes only have acid-producing functions
.

 Figure 3 shows the abundance changes of metabolically active dominant functional bacteria in the anaerobic digestion reactor at different operating stages
.

It was found that the total abundance of amino acid-degrading bacteria showed a gradual downward trend throughout the operation process, and a 31.
86% decrease in stage I2 (Fig.
3b); the total abundance of propionic acid oxidizing bacteria (SPOB) did not change significantly in stage I1.
, then increased significantly in the recovery period (p < 0.
05), but decreased by 51.
79% in the I2 stage (p < 0.
05), even significantly lower than the stable and I1 stages (p < 0.
05) (Fig.
3d)
.

Besides, the abundance of active microorganisms in other functional flora gradually increased or remained stable during the operation (Fig.
3a,c,e,f)
.

The indicator amino acid degrading bacteria and SPOB may be the key functional groups of potential destabilization
.

Figure 4.
Number of differentially expressed proteins (DEPs) (ae) and key enzyme expression information (f) of active functional bacteria
.

Positive numbers in ae represent the number of up-regulated proteins, and negative numbers represent the number of down-regulated proteins
.

In f, yellow indicates significantly up-regulated expression, blue indicates significantly down-regulated expression, and "_" indicates that the protein is only present in one of the two groups of samples being compared
.

 Figure 4 further analyzes the changes in functional protein expression profiles of potentially key functional bacteria
.

Considering the accumulation of VFAs in the destabilizing stage, in addition to amino acid-degrading bacteria and SPOB, the functional protein expression profiles of the mutual-accepting acetate, butyrate, and valeric acid-degrading bacteria and methanogens related to VFAs metabolism were also analyzed (Fig.
4)
.

The results showed that: (1) Although the amino acid metabolism function of the system was damaged in the I2 stage, the presence of highly tolerant and functionally redundant microorganisms such as Aminobacterium is expected to restore the amino acid metabolism performance after long-term domestication, and amino acid metabolism may not lead to the destabilization of the reactor.
the root
of .

(2) In the I1 stage, ammonia nitrogen mainly caused its acetate-type methanogenesis (AM) function to be impaired by inhibiting the expression of methyl-CoM reductase of Methanothrix.
The metabolic function was completely restored, and the acetic acid metabolism was not the source of the instability of the reactor
.

(3) In stage I2, ammonia nitrogen inhibited the metabolic activity of the genus Methylmalonyl-CoA Pathway (MMC pathway) by inhibiting the expression of succinyl-CoA synthase that dominates SPOB (Pelotomaculum), resulting in the repression of propionic acid metabolism in the system, the substantial accumulation of propionic acid, and the rest The abundance of active SPOB also decreased significantly in the I2 stage, therefore, propionic acid oxidizing bacteria may be the key microorganisms causing the instability of the reactor
.

Figure 5.
The mechanism of ammonia inhibition and instability in the anaerobic digestion system is comprehensively analyzed by physical and chemical analysis and microbial analysis (Figure 5).
Acetate metabolism is most sensitive to ammonia stress.
Methanothrix is ​​inhibited at a relatively low ammonia nitrogen concentration, resulting in the accumulation of acetic acid in the system.
And through the feedback effect caused the accumulation of other VFAs
.

But at this time the community as a whole was robust, and with the extension of the run time, the functionally redundant Methanosarcina were strengthened, and they restored the acetate metabolism performance through the metabolic pathways mediated by acetatekinase, phosphate acetyltransferase and ACDS
.

This indicates that acetate metabolism is not the key link of ammonia inhibition and instability, and the community can adapt to this level of ammonia stress environment
.

However, with the continuous accumulation of ammonia nitrogen, propionic acid alone accumulated in the system, and the gas production again decreased by more than 50%
.

At this time, the abundance of active SPOB decreased by nearly 50% and the expression of differential proteins was down-regulated by 94%, indicating that this type of functional bacteria was severely inhibited and difficult to recover
.

Therefore, SPOB is a key functional bacterium that affects the performance of anaerobic digestion under ammonia stress
.

High ammonia stress limits its growth and metabolism mainly by inhibiting the expression of its succinyl-CoA synthase, blocking the MMC pathway, resulting in propionic acid accumulation and system instability
.

Summary Ammonia inhibition is one of the most common factors contributing to anaerobic digester instability
.

This study combines metagenomics and metaproteomics to investigate anaerobic digester performance and microecological responses during endogenous ammonia accumulation
.

The results showed that among AD metabolic segments, acetate metabolism was most sensitive to ammonia nitrogen, and lower ammonia nitrogen concentration would inhibit the expression of methyl-CoM reductase of Methanothrix and inhibit its acetate metabolism function; however, continuous ammonia stress could degrade the dominant acetate.
The bacteria were converted from Methanothrix to Methanosarcina, which restored the systemic acetic acid metabolism
.

In contrast, the link of propionate metabolism is the key link leading to the destabilization of ammonia inhibition.
High ammonia nitrogen will inhibit the expression of succinyl-CoA synthase in Pelotomaculum to suppress propionate metabolism
.

In the later stage, more attention should be paid to the SPO process for the microbial regulation of ammonia-inhibited anaerobic digestion reactors
.

About the main author The first author: Zhang Hong, a 2021 doctoral student at the School of Environment and Ecology, Chongqing University, whose research direction is solid waste pollution control and recycling
.

Corresponding author: Li Lei, female, Ph.
D.
, associate professor/doctoral supervisor
.

The research direction is solid waste pollution control and resource utilization
.

He has successively presided over national, provincial and ministerial-level projects such as general projects of the National Natural Science Foundation of China, Youth Fund, sub-projects of the National Key R&D Program, Chongqing Basic Research and Frontier Exploration, and Central University Funds; completed the ecological restoration of landfills, Several engineering projects such as pollution control
.

Published more than 30 papers in top international and domestic journals such as Water Res.
, Renew.
Sust.
Energ.
Rev.
, Bioresour.
Technol.
, Sci.
Total Environ.
; applied for 5 national invention patents; won the first prize of Chongqing Science and Technology Progress Award 1 item
.

Email: lileich17@cqu.
edu.
cn Team leader: Peng Xuya, male, Ph.
D.
, professor/doctoral supervisor, has long been engaged in theoretical and technical research on solid waste pollution control and recycling
.

He has presided over the main research and completed more than 40 national and provincial (ministerial) research projects (including the National Natural Science Foundation of China, the National Water Project, the National Science and Technology Support (Tackling) Program, International Cooperation Projects, Ministry of Agriculture and Chongqing Municipal Major Science and Technology Projects) etc.
), presided over and completed more than 60 horizontal research projects
.

Participated in the compilation of 9 national and Chongqing local standards; participated in the compilation and publication of 1 monograph
.

Obtained 11 national invention patents
.

He has published more than 150 papers in domestic and foreign academic journals
.

He has won 1 first prize of Huaxia Construction Science and Technology Award, 1 second prize of Science and Technology Progress Award of the Ministry of Education, and 1 first prize and second prize of Chongqing Science and Technology Progress Award
.

Contribution: Peng Xuya/Li Lei team of Chongqing University
.

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