Indigenous cellulolytic aerobic and facultative anaerobic bacterial community enhanced the composting of rice straw and chicken manure with biochar addition

Composting characteristics

Table 2 shows the main characteristics of the composting process with and without the addition of biochar. The final composting process with biochar showed a significant (P<0.05) reduction of carbon, representing 19% of the initial C content, whereas in the composting without biochar, carbon reduction was only 8%. Both piles showed the escalation of the N content at the final stage of the composting process. However, the addition of biochar did not show any significant reduction (P>0.05) in the amount of N, supporting the hypothesis that biochar retains nitrogen in the composting mixture through the absorption of the nitrogenous compounds such as ammonia and ammonium onto the surface of biochar. Previous research has shown that composting with biochar reduces NH₃ emissions while increasing NO₃^– concentrations when compared to composting without biochar. According to Zainudin et al.¹³, the biochar addition to the composting pile improves the nitrification process, in which NH₄⁺ is transformed into NO₃^– by nitrifying bacteria. Furthermore, the oxidation of aromatic and carbonyl groups led to the creation of a positive and negative charge on the surface of biochar, promoting NH₄⁺ and NO₃^– adsorption¹⁸. The overall decrease in C content was higher in the biochar treatment pile than in the composting without biochar. In this study, composting was conducted by combining the chicken manure and rice straw at the ratio of 20:1 ratio, or C/N:15.6. According to Zhu¹⁹ and Zhou²⁰, aerobic composting of swine manure with rice straw, edible fungal residue and rice bran at a low C/N ratio improves the maturation rate and increases organic matter degradation as compared to high C/N ratio composting. This finding is also in accordance with a previous study, which found that the dissolved organic carbon (DOC) was lower in biochar amended compost than without biochar due to the increased microbial activity¹⁸. It has been suggested in previous studies that adding biochar reduces the bulk density, which subsequently improves the aeration of the composting pile. The improved aeration promotes microbial proliferation and activity,thus, enhancing nutrient mineralization throughout the composting process. Thus, our finding supports the evidence of previous studies which reported that biochar improves organic matter degradation, hence, reducing the carbon content. Aside from the higher C reduction, the macro- and micronutrients such as phosphorus, kalium, calcium, magnesium, and zinc in composting with biochar were also higher than that of control composting. This could be due to the higher inorganic nutrient contents in the EFB biochar^14,15. In addition, the organic coating forms on the outer and inner pore surfaces of biochar particles promotes the nutrient retention of the co-composted biochar²¹. The temperature of the composting process lasted for 14 days in control composting and 18 days in composting with biochar (Fig. 1a). The extended thermophilic phase is common for this organic material because rice straw contains recalcitrant compounds that are difficult for microorganisms to degrade, thus lengthening the composting process. However, the maximum temperature of the pile was higher in composting with biochar (74 °C) than in composting without biochar (62 °C), indicating that the addition of biochar enhanced the microbial activities, especially for the lignocellulose degradation. A study conducted by Huang et al.²² indicated that composting of pig manure and sawdust with an initial C/N of 15 resulted in a gradual rise in temperature, lower maximum temperature, and shorter thermophilic phase. However, it is interesting to demonstrate in our study that higher maximum temperature, rapid temperature increase, and longer thermophilic period can be achieved in composting with biochar at a low C/N ratio, implying the function of biochar in promoting the composting process as a result of higher organic matter degradation by microbes.

Profiles of temperature (a), pH (b) and moisture content (c) throughout the composting process with and without biochar addition. The blue arrows indicate the turning of composting pile. The data represent the average of replicated samples. The temperature was recorded by inserting the probe horizontally into the core of the compost pile (90 cm) at 3 different positions (upper, middle and lower). The pH was recorded from the combined sample taken from different sampling points (middle and bottom part of compost pile).

The pH of the pile increased, whereas the moisture decreased as the composting progressed (Fig.1b,c). The results corresponded with a previous study which reported that the biochar addition increased the pH of the composting pile, which was due to the alkalinity properties of the biochar²³. The physicochemical properties suggest that the enhancement of composting process could be due to the addition of biochar which improves the microbial activities for organic matter degradation. Therefore, in this study, we attempt to identify the bacterial community structure using high-throughput 16S rRNA gene sequencing and isolation of cellulolytic bacteria to evaluate further their relevance in improving the composting process with biochar.

Bacterial community structure

The high-throughput 16S rRNA gene sequencing was performed to clarify detailed information about the bacterial population during both of the composting processes. The heatmap analysis showed that the composting with biochar was generally dominated by the Thermobifida, Nocardiopsis, Compostibacillus, Ammonibacillus, Sinibacillus, Bacillus, Truepera, Halomonas, Pseudofulvimonas, respectively (Fig. 2). These bacteria were often discovered during agricultural waste composting^13,24,25,26. During the thermophilic phase, the most prevalent taxa were Sinibacillus, Bacillus, Compostibacillus and Thermobifida. However, the abundance of Sinibacillus, Bacillus, and Thermobifida decreased as the composting progressed. In contrast, the abundance of Nocardiopsis increases when the composting enters the mesophilic and mature phases. Thermobifida and Nocardiopsis were highly abundant in the composting with biochar as compared to composting without biochar.

Heatmap analysis of the bacterial genera with the relative abundance of ≥ 0.1%. The (B) label denotes composting with biochar, while the (C) label denotes composting without biochar.

In addition, the abundance of Bacillus, Sinibacillus and Compostibacillus during the thermophilic phase of the composting with biochar was higher than that of composting without biochar. These bacteria were able to live at the high temperature of the composting pile due to their ability to generate endospores. A previous study showed that members of Bacillus and Sinibacillus were mainly responsible for the degradation of high-molecular-weight of organic matter throughout the composting process²⁴. Moreover, Compostibacillus which was previously isolated from sludge composting has been found as a facultative anaerobe and is capable of growing at temperatures as high as 60 °C²⁷. Facultative anaerobes can grow and metabolize in aerobic and anaerobic environments although they favor oxygen-rich conditions²⁸. Therefore, the increased abundance of Compostibacillus supports the finding of previous studies that the addition of biochar enhanced the aeration of the composting pile, thus, encouraging the development of bacteria including facultative anaerobe. Truepera, Halomonas and Pseudofulvimonas genera which are known as nitrifying and denitrifying bacteria¹³ showed an increasing trend as the composting entered the mesophilic and maturing stages. These bacteria were found to be highly dominant in the composting with biochar.

Cellulolytic bacteria

Composting with biochar resulted in a greater reduction of carbon content, which might have been assisted by the presence of cellulolytic bacteria. As a result, in this study, we aim to isolate as many cellulolytic bacteria as possible from the composting with biochar. The isolation of cellulolytic bacteria was also done from the composting without biochar for comparison. The results showed that the number of isolated cellulolytic bacteria was greater in composting with biochar than in composting without biochar (Table 3). Twenty-eight of the isolated strains were found to be more than 99% identical to known cellulolytic bacteria. These strains were closely related to Bacillus licheniformis, Bacillus subtilis, Bacillus aerius, Bacillus haynesii, all of which were known to exhibit cellulolytic activity and have been involved in the lignocellulose degradation process^29,30,31. Of these cellulolytic strains, B. licheniformis was the primary species isolated from the composting with biochar. The results corresponded with 16S rRNA sequencing data, which indicated that Bacillus was among the dominant bacteria detected throughout the composting process. B. subtilis had previously been isolated from soil and compost and was known to demonstrate cellulolytic activities^32,33.

This bacterium has been widely used in various kinds of applications including cellulase production for saccharification and as an inoculum to enhance the composting process^34,35. B. aerius is a thermophilic cellulolytic bacterium that was previously isolated from lignocellulosic waste and hot-spring sediment^30,36. This bacterium plays a major role in the hydrolysis of lignocellulose and has been found to produce highly thermostable cellulases³⁷.

Relationships between physicochemical properties and bacterial community

Since composting is a microbially-mediated process, understanding the relationship between microbe and physicochemical properties of the compost will substantially improve the efficiency of the composting process and the quality of its end-product. Therefore, a principal component analysis (PCA) was performed to explain the relationship between the dominant bacteria community, particularly cellulolytic bacteria, and physicochemical characteristics during the composting process with biochar. The results showed that Thermobifida and Nocardiopsis exhibited positive correlations with N content and pH, respectively (Fig. 3). In contrast, they showed negative correlations with C content, indicating their important roles in organic matters degradation, particularly the lignocellulosic materials, thus, reducing the C content as the composting progressed. Thermobifida and Nocardiopsis genera were known as cellulolytic bacteria and had been involved in the lignocellulose degradation^17,25. They were important cellulolytic actinobacteria capable of generating a variety of hydrolytic enzymes for lignocellulose degradation, including exoglucanase, endoglucanase, β-glucosidase and xylanase. The majority of the enzymes produced by these bacteria were very stable at a broad range of pH and temperature^38,39. These bacteria are aerobic, gram-positive and spore-forming bacteria and these traits are critical for their survival in harsh environments.

Principal component analysis (PCA) indicates the correlation and distribution of the samples between dominant phyla and physicochemical parameters during the composting with biochar. Interpretation of the data analysis was explained by Biplot. Original variables (dominant phyla and physicochemical characteristics) drawn as vectors (red line) was used to summarize the correlation between the variable. Clustering of the sample site due to the bacteria community composition and composting stages are highlighted by green, brown, and dark red dots. F1 and F2 axes represent 62.3% of the explained variance.

Similar to Thermobifida and Nocardiopsis, the cellulolytic B.licheniformis, also showed an inverse correlation with the C content, indicating its function in the degradation of the lignocellulose matrix. On the other hand, B.licheniformis positively correlated with pH but negatively correlated with moisture content and temperature. B. licheniformis is a facultatively anaerobic, gram-positive bacterium that has been isolated in a wide range of temperature environments⁴⁰. In this study, B. licheniformis was successfully isolated at mesophilic and thermophilic temperatures, indicating that this bacterium can grow and thrive in both conditions⁴¹. According to He et al.⁴², B. licheniformis would grow fast and require less doubling time in the presence of oxygen. As suggested earlier, the purpose of adding biochar into the composting mixture was to lower the bulk density of the components, hence, enhancing the oxygen penetration to the interior part of the pile. The high-water content of the composting without biochar (Fig. 1c) might saturate the system and reduce the oxygen penetration, thus, decreasing the B. licheniformis population. As a result, the reduced moisture content of the composting with biochar enhanced the compost environment for the B. licheniformis development. Previous studies reported that the micro-aerobic pretreatment of lignocellulose corn straw and microalgal biomass increased the activity of the hydrolytic enzymes such as cellulase and encouraged the development of facultative anaerobic bacteria^42,43. Furthermore, B. licheniformis isolated from bovine rumen had the highest cellulose-degrading activity under micro-aerophilic conditions⁴⁴. This corresponds with our earlier discussion that the increasing number of cellulolytic facultative anaerobe B. licheniformis could be attributed primarily to adequate oxygen supplementation for this bacterium to thrive as a result of the large surface area and porosity of biochar, which produces the micro-aerobic conditions inside of the composting pile. In addition, Truepera, Halomonas, Pseudofulvimonas also showed a negative correlation with C content, indicating that the increased abundance of these genera could be attributed to the consumption of readily utilizable organic fraction generated as a result of the organic matter degradation during the thermophilic stages, hence, reducing the carbon content towards the end of the composting process. Overall, the findings of this study suggest that the enhancement of the composting with biochar was due to the abundance of cellulolytic bacteria detected, which may have led to higher organic matter degradation especially the lignocellulosic material throughout the composting process.