Community succession and functional prediction of microbial consortium with straw degradation during subculture at low temperature

Changes of straw degradation characteristics at different culture stages

Corn straw degradation ratio

Corn straw weight loss in M44 at F1 reached 35.90% at 15 ℃ for 21 days, which was greater than that at F5, F8, and F11 by 2.33%, 3.01%, and 3.35%, respectively. There were no significant differences between F8 and F11(Fig. 1).

Enzyme activities

The highest endoglucanase and filter paper enzyme activities were 2.01 and 2.16 U mL⁻¹ in F1, respectively, which was significantly higher than that of F8 and F11(Fig. 2a,b). Xylanase activities was the highest in F5, with enzyme activities of 21.50 U mL⁻¹, and the enzyme activities of F1 and F5 were significantly higher than those of F8 and F11(Fig. 2c). Laccase and lignin peroxidase activity reached 101.02 and 80.37 U L⁻¹ at F5, which was greatly different than that of other algebras (Fig. 2d,e).

VFA content

Acetic acid, prophetic acid, and butyric acid contents were all the highest at F5. Acetic acid and propionic acid contents were 143.91 mmol L⁻¹ and 6.70 mmol L⁻¹ at F5, respectively, which were significantly different from those at F1 and F8 (Fig. 3a,b). Butyric acid was 3.80 mmol L⁻¹ in F5, which was significantly different from that in F1 but not from that in F8 and F11(Fig. 3c).

Alpha diversity of microorganisms at different culture stages

Alpha diversity was used as a measure of microbial community diversity within the sample. The Ace and Sobs indexes for the original samples(S) were 1358.22 and 1101.33, respectively, which were significantly higher than those of F1, F5, F8, F11. Shannon and Simpson indexes showed the opposite trend, with values of 2.59 and 0.161 in the F5 generation, respectively, indicating that the microbial community was more abundant and diverse in this culture stage (Fig. 4).

Beta diversity of microorganisms at different culture stages

A principal component analysis (PCA) was conducted on the bacterial community in each group of samples, and the results was shown in Fig. 5. The contribution rates of PC1 and PC2 were 42.64% and 23.3% of the total, respectively. Samples of S and F1, F8, and F11 clustered together, indicating that the composition of microbial communities in these two groups was similar. On the other hand, samples from F5 clustered far from each other and into a single cluster, indicating that the microbial community compositions of the F5 samples were significantly different from those of other periods. To further define the differences, ANOSIM and PERMANOVA were performed at the OTU level based on the Bray–Curtis distance algorithm. The results showed that there were significant differences between different stages (p < 0.05; N = 999 permutations).

Taxonomic composition analyses at different culture stages

Phylum level

The relative abundance of bacterial groups according to classification level was shown in Fig. 6. At the phylum level, microbial consortium M44 was mainly composed of Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Verrucomicrobia. Among these, Proteobacteria was dominant in M44, with abundances of 56.84%, 87.09%, 61.64%, and 53.94% at F1, F5, F8, and F11, respectively. Bacteroidota accounted for 32.11% of the total bacterial content in the F1, which was significantly higher than that of S, F5, F8, F11. The relative abundance of Firmicutes increased steadily, accounting for 26.80% of the total in F11, which was considerably higher than that at F1 (5.83%), F5 (7.68%), and F8 (17.37%). The relative abundance of Actinobacteriota in the original sample(S) was 41.19%, but decreased with subculture of the microbial consortium. The relative abundance of Verrucomicrobiota fluctuated with increasing culture stage, increasing to 3.03% in F11.

Genus level

At the genus level (Fig. 7), the relative abundance of Pseudomonas was 0.46% in the original sample(S), with its abundance was shown increased first and decreased then with culture time, reaching 8.75% in F11. The relative abundance of Brevundimonas was highest in the F1, was 10.79%, and which was significantly different from that in F5, F8 and F11. The relative abundance of Flavobacteria in the original sample(S) was 0.53%, which increased first and decreased then across culture stages. The relative abundance of Devosia was 3.09%, with its highest abundance at 1.71% in the F1 generation, after decreased with time. The relative abundances of Achromobacter and Ochrobactrum in F1 were 5.60% and 6.56%, respectively, but bacteria in these genera were rarely found in the original samples(S). Their relative abundances decreased with increasing culture time. In addition, Trichococcus, Acinetobacter and Azospirillum were found in F5, and the relative abundances of F11 were 19.65%, 13.01%, and 2.96%, respectively.

Correlation analysis of microbial community at different culture stages

The analysis of the dominant microbial community in the microbial consortium at different culture periods showed (Fig. 8) that Proteobacteria contained the largest number of genera with relatively distant evolution, and was mainly composed of 12 genera, including Pseudomonas, Azospirillum, Brevundimonas and Ochrobactrum. Among them, the abundance of Pseudomonas was dominant in the F1, F5, F8 and F11 generation samples, which played a key role in the degradation process of straw. Bacteroidetes was composed of Flavobacterium, Dysgonomonas and Taibaiella with similar evolution. It could be concluded that Proteobacteria was the main functional bacteria involved in straw degradation in samples of different culture periods.

The correlation network diagram was used to study the interrelationship between straw degrading microorganisms of M44 in different subculture periods (Fig. 9). Rhizobium, Acinetobacter, Trichococcus, Dysgonomonas, Azospirillum, Enterobacter were strongly correlated with each other and positively correlated with other bacteria. The results showed that there were significant interactions among different genera in the samples of different culture periods, and a variety of microorganisms synergistically degraded corn straw.

Correlation analyses of physicochemical characteristics and dominant genera

Correlation analysis between the TOP20 genera in M44 and straw degradation characteristics (Fig. 10) showed that endoglucanase activity was positively correlated with Brevundimonas, Achromobacter, Hydrogenophaga, Chryseobacterium, Sphingobacterium, and some bacteria that degrade lignocellulosic or intermediate products in the colony. Dysgonomonas had a significant negative correlation with filter paper enzyme activity, Acinetobacter had a significant negative correlation with xylanase activity, and Pseudomonas and Enterobacter had a significant positive correlation with laccase and lignin peroxidase activity. Rhizobium and Proteiniphilum were positively correlated with acetic acid, prophetic acid, and butyric acid contents.

Functional prediction analysis

The COG database comparison

Based on COG database comparison results (Fig. 11), it was found that the function of the M44 was mainly concentrated Amino acid transport and metabolism, General functional prediction only, Transcription, Carbohydrate transport and metabolism and Cell wall/membrane/envelope biogenesis and so on in different culture stages. It could be predicted that M44 may contain abundant genes related to protein decomposition, transport and metabolism enzymes, as well as a large number of genes related to cellulose and lignin degradation enzymes during subculture at low temperature.

The KEGG database comparison

According to KEGG level 1 (Table 1), genes in samples of different culture stages were mainly enriched in Metabolism, Environmental Information Processing, Genetic Information Processing, Cellular Processes, etc. Among them, Metabolism accounted for the highest proportion, and the proportion of original samples was 78.91%, which showed no significant difference with F1, F5, F8 and F11 generation samples. There were 46 metabolic pathways in KEGG Level 2. Table 2 showed the results of the top 15 pathway abundance values in different culture stages. The main metabolic pathways included Global and overview maps, Carbohydrate metabolism, Amino acid metabolism, Energy metabolism, Metabolism of cofactors and vitamins, Membrane transport and Signal transduction, etc. The top 30 enzymes in abundance were further analyzed (Table 3), the results showed that the relative abundance of DNA-directed DNA polymerase, DNA helicase, Peptidylprolyl isomerase, NADH:ubiquinone reductase (H( +)-translocating), 3-oxoacyl-[acyl-carrier-protein] reductase were high. In addition, the Peroxiredoxin, Acetyl-CoA carboxylase were present in different culture periods, and the abundance is obviously different.

Source: Ecology - nature.com