Effects of Inoculation of Thermotolerant Bacillus Strains on Lignocellulose Degradation

Abstract

Thise study investigated the effect of three lignocellulolytic thermophilic Bacillus strains (F11, Q1, and FP4) on lignocellulose degradation, enzymatic activities, and microbial community structure in composting. The lignin degradation rate reached 36% in the presence of the inoculant, the hemicellulose degradation rate ranged from 43% (F11) to 51% (Q1), and cellulose degradation rates reached 57% in F11 and in FP4, which were significantly higher than the control (CK). The inoculation treatment could explain 28% of the lignin degradation for all three strains. The contribution of FP4 to hemicellulose and cellulose degradation was 30% and 20%, respectively. Compared to CK, lignin peroxidase activity in the water extract of the compost had increased by 66~145% for inoculation treatments, and manganese peroxidase and laccase activity increased by 114% and 78% for Q1. The inoculation stimulated the growth of indigenous bacteria with stronger lignocellulolytic enzyme-producing ability; such shifts in microbial communities were most likely responsible for the improved lignocellulose degradation.

1. Introduction

Agricultural wastes primarily come from crop residues; tomatoes and muskmelons are common crops with high yields that generate a large amount of residues after harvest. Biodegradable components of agricultural wastes are degraded by microbial communities during composting. The obtained compost contains valuable nutrients and organic matter that can be added back to soils, improving soil structure and water-holding capacities. When composting is performed at a high enough temperature, pathogenic bacteria that might be present in the original plant material can be inactivated. This holds significant practical importance for promoting sustainable agricultural development.

Plant material typically contains lignocellulose, a complex macromolecular structure consisting of lignin, hemicellulose, and cellulose. Lignin is a three-dimensional polymer network composed of aromatic phenylpropane units connected by ether and carbon-carbon bonds, and this is covalently bound to hemicellulosic side chains. Cellulose is a homopolymer consisting of a linear chain of several hundred to many thousands of β-anhydroglucose units (β-1,4 linked d-glucose units). Hemicellulose is a heteropolymer consisting of a polysaccharide backbone. Its structure greatly varies depending on the sugar units, chain length, and the branching of the chain molecules [1]. Lignocellulose usually amounts to the majority fraction of the total organic matter in vegetable residues. It is resilient to degradation by microbes, and its conversion rate during aerobic composting by endogenous microorganisms is relatively low [2,3]. Microbes play a key role in composting; inoculation with exogenous microbes can improve lignocellulose degradation, and this has been the subject of a lot of research [4,5,6]. Such work has identified the key enzymes released by microorganisms during composting [7,8,9]. Lignocellulose degradation is enabled by enzymes secreted by microorganisms, mainly ligninase, hemicellulase, and cellulase, whose action is synergistic [7]. Composting is an attractive method for disposing of agricultural organic residue that involves the breaking down of biodegradable components through the action of bacteria, actinomycetal, and fungal [10]. This process encompasses 3 primary stages. Initially, there is the decomposition phase, characterized by temperatures hovering between 30 and 50 °C. During this phase, mesophilic microorganisms rapidly decompose readily biodegradable organic matter, a process that typically spans from a few days to a couple of weeks. Subsequently, the thermophilic phase takes center stage, with temperatures escalating to between 50 and 70 °C, as the decomposition of organic matter continues. This phase is predominantly governed by thermophilic microorganisms capable of decomposing more intricate organic materials like cellulose and lignin. Lastly, the maturation phase unfolds as the abundance of easily degradable materials diminishes, resulting in a decline in temperature and a reduction in microbial activity. During this final stage, microorganisms persist in breaking down more resilient organic matter and initiate the formation of stable humus [11]. Composting can result in high temperatures, which may unfavorably inhibit the growth of the inoculated organisms [6,12]. Elevated temperatures may also remove the majority of fungal populations in favor of Actinomycetes, a shift that can delay the decomposition of organic matter. This would suggest that bioaugmentation of composting is best performed with thermotolerant strains that produce lignocellulosic degrading enzymes. A number of useful thermophilic lignocellulose-degrading bacteria have been described, such as Bacillus, Bacillus subtilis, and Brevibacillus thermoruber B. [13,14]. These are typically abundant in composting, especially during states with elevated temperatures [15]. They frequently produce lignocellulose-degrading enzymes [16] and can resist harsh environments. Therefore, Bacillus inoculants might be good candidates for efficient lignocellulose breakdown during composting.

In preparative work, we collected bacterial samples from a mixture of tomato and melon plant wastes during the thermophilic stage of composting. These isolates were primarily screened, purified, and rescreened to obtain strains of thermotolerant lignin-degrading Bacillus members. Three strains, designated F11, FP4, and Q1, were considered suitable to test the following hypotheses: (1) inoculation with a thermotolerant strain of Bacillus sp. might contribute to higher lignocellulose degradation rates by improving lignocellulase activity during composting, and (2) such inoculation affects the microbial population structure and increases the relative abundance of Bacillus. The detailed objectives of this study were as follows: (I) to detect the influence of strains F11, Q1, and FP4 inoculation on the degradation of lignocellulose; (II) to evaluate the effect of such inoculation on specific enzyme activity; and (III) to analyze the microbial population structure. The aim of this study was to determine the effect of inoculation with the three strains on lignocellulose biodegradation as well as on the microbial structure of the compost. This study provides a theoretical basis for the inoculation approach to promote the degradation and conversion of recalcitrant lignocellulose during composting.

2. Materials and Methods

2.1. Bacterial Strains and Composting Material

The three thermotolerant strains of Bacillus sp. used in this study, designated F11, Q1, and FP4, were isolated from thermal composting of tomato and muskmelon residues (1:1, w:w). The strains were selected based on their thermo-resistance which was tested for temperatures up to 70 °C. Strains F11, Q1, and FP4 could resist temperatures up to 50 °C, 55 °C, and 70 °C, respectively. During screening, their ability to decompose lignin was assessed by the Bavendamn method and verified by ligninolytic enzyme activities during liquid fermentation [17]. To prepare inoculants, the strains were cultivated in NB medium (beef extract 5 g; peptone 15 g; NaCl 5 g; agar 20 g; water 1 L; pH 7.0.) at 50 °C, with shaking at 150 rpm/min for 48 h. The liquid cultures were collected, and their volume was adjusted to 1.5 × 10⁸ CFU mL⁻¹.

2.2. Composting Experiment Design and Sampling

The materials used for composting are the stems and leaves of tomatoes and muskmelons collected from a vegetable farm. Before composting, the residues were chopped into 1–2 cm pieces and mixed evenly. This resulted in a mixture containing 7.5% lignin, 19% hemicellulose, and 19% cellulose. The TOC content was 422 g/kg, the total N content was 33.5 g/kg, the C/N ratio was 19:1, and the mixture had a moisture content of 68%. The content of lignin, hemicellulose, or cellulose was determined by the improved Van Soest method [18,19]. Toc, nitrogen, and moisture content were determined according to the standard methods for organic fertilizers [20].

Composting experiments were carried out in a temperature-controlled glasshouse with a daily average temperature varying from 22 °C to 28 °C. Twelve laboratory-scale bin reactors with a content of 21 L were used (sized 27 cm × 27 cm × 42 cm), with bars at the bottom and holes at the top that enabled ventilation. The bars were filled with 5 kg of composting material, and triplicates were inoculated with 100 mL of liquid bacterial culture of strain F11, strain Q1, or strain FP4, respectively. Inoculation was repeated with the same strain after 7 days of composting. A control (CK) treatment received water only, also performed in triplicate. The composting materials were mixed thoroughly following inoculation and then left standing for 8 days to give a total composting duration of 15 days.

At the end of the experiment, samples were collected from three locations within each bin: one from the surface, one from the central part, and one from the bottom. These three samples were thoroughly mixed and split into 3 fractions. One fraction was air-dried at 40 °C prior to lignocellulose determination, the second was immediately used to measure enzymatic activity, and the third was used for microbial analysis.

2.3. Determination of Lignocellulose Degradation

The content of lignin, hemicellulose, or cellulose was determined by the improved Van Soest method [18,19]. The hemicellulose content was estimated as the difference between neutral-detergent fiber (NDF) and acid-detergent fiber (ADF), and the cellulose as the difference between ADF and acid-detergent lignin (ADL). The content of lignin was estimated as the difference between ADL and ash content. From this, the lignocellulose content was calculated.

The contents of lignin, hemicellulose, and cellulose were determined, from which the overall degradation and treatment degradation rates were calculated:

Overall degradation rate (%) = [(X₀ − X₁₅)/W₀] × 100%

(1)

where X₀ is initial content at day zero of lignin, hemicellulose, or cellulose, X₁₅ is content of lignin, hemicellulose, or cellulose at day 15 after composition, and W₀ is initial composting material weight.

Treatment degradation rate (%) = [(X₁₅(ck) − X₁₅(tr)/X₁₅(ck)] × 100%

(2)

where X₁₅(ck) is the amount of lignin, hemicellulose, or cellulose at day 15 in the control and X₁₅(tr) is the amount of lignin, hemicellulose, or cellulose at day 15 in the treatment. The treatment degradation rate specifies the contribution of the treatment to the overall degradation.

2.4. Determination of Lignocellulose-Degrading Enzyme Activities

Fresh compost samples (5 g) were extracted with 50 mL of distilled water under rotary shaking (200 rpm) for 1 h, then left to set, and 5 mL of the liquid phase was centrifuged at 4 °C, 10,000 rpm for 10 min. The obtained supernatant (crude enzyme extract) was used to determine xylanase and carboxymethyl cellulase (CMC) activity according to the methods described before [21]. One unit (U) of CMCase or xylanase activity was defined as the amount of enzyme required to produce 1 μg of glucose or xylose per minute.

Fresh compost samples (5 g) were extracted with 100 mL of distilled water under rotary shaking (150 rpm) for 1 h, then centrifuged at 4 °C, 10,000 rpm for 10 min. The obtained supernatant was used to determine the activity of lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase activity (Lac), using the methods described by [8]. For these enzymes, 1 unit was defined as the amount of enzyme that led to the production of 1 μmol product per minute (veratryl aldehyde for LiP, Mn³⁺ per for CMC, 2,2-azino-bis-[3-ethyltiazoline-6-sulfonate] (ABTS) for laccase [8].

2.5. Microbial Community Composition and Diversity Analysis

High-throughput sequencing of partial 16S rRNA amplicons was applied to determine the bacteria diversity in the compost, and primers targeting fungal 18S rRNA were used to determine the fungal diversity.

DNA isolation, PCR amplification, Miseq sequencing, and processing of sequencing data were performed using the Illumina Miseq platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China. The methodology was followed as described in [22,23]. The data were analyzed on the online platform of Majorbio Cloud Platform (www.majorbio.com, accessed on 6 May 2021). Operational taxonomic units (OTUs) with a 97% similarity cutoff were clustered using UPARSE version 7.1, and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 against the 16S rRNA database (Silva v138) and the 18S rRNA (PR2 v1.0). Community richness parameters (Chao1, ACE), and community diversity parameters (Shannon, Simpson) were calculated using the Mothu software, version 1.30.2 (https://www.mothur.org/wiki/Download_mothur, accessed on 6 May 2021). Beta diversity measurements, including approximately maximum likelihood phylogenetic trees mapped using FastTree, version 2.1.11 (http://www.microbesonline.org/fasttree/, accessed on 6 May 2021) as well as principal coordinate analyses (PCoA) based on OUT compositions, were calculated.

The differences in these indices among different groups were tested by Duncan’s multiple comparisons. Weighted unifrac distances were calculated with GUniFrac package and principal coordinate analysis (PCoA) was performed using ape package. The significant differences in communities among different groups were identified by permutational multivariate analysis of variance (PERMANOVA) based on the Bray-Curtis dissimilarity matrix.

3. Results and Discussion

3.1. Effect of Inoculants on the Overall Lignocellulose Degradation Rate

The degradation of lignin, hemicellulose, and cellulose is crucial for the composting process. Following experimental composting of tomato/melon plant waste in the presence of inoculant F11, Q1, or FP4 and without inoculant (CK) for 15 days, the overall degradation rates of the three lignocellulose components lignin, hemicellulose, and cellulose were determined (Figure 1A). The degradation rates of lignin were less than those of cellulose and hemicellulose lignin [24]. Observed a similar phenomenon, which was attributed to the fact that the molecular weight of lignin is significantly higher than that of cellulose and hemicellulose, as well as its complex chemical structure and strong polarity. All three overall degradation rates were higher for treatment in the presence of the inoculants compared to CK. Cellulose and hemicellulose were subjected to higher degradation rates than lignin, a trend that was also observed by others [7]. Lignin degradation rates in the presence of F11, Q1, and FP4 were all identical (36%), which was significantly higher than the control (29%). The overall degradation rate of hemicellulose varied between F11, Q1, and FP4 (43%, 51%, and 49%, respectively); however, the difference between the three inoculant treatments was not significant. Inoculant Q1 degraded most hemicellulose, and this resulted in a 25% better hemicellulose breakdown than CK. Similarly, the cellulose degradation rate did not significantly differ between the three inoculants, but both F11 and FP4 reached rates of 57%, which was approximately 10% higher than that of CK.

The treatment degradation rate that could be attributed to the effect of the inoculant was calculated after correction for the degradation observed in CK (Figure 1B). The treatment lignin degradation rate was the same (10%) for all three inoculants, and inoculation was responsible for 28% of the overall lignin degradation. The treatment hemicellulose degradation rate of F11 was below 5%, but Q1 and FP4 ( 17% and 15%, respectively) contributed 34% and 30%, respectively, to overall hemicellulose degradation. The treatment cellulose degradation rate of F11 and FP4 was 11%, giving a contribution of 20% of the overall cellulose degradation; the contribution of Q1 was slightly less. Thus, strain Q1 resulted in more effective degradation of hemicellulose, but strains F11 and FP4 contributed more to cellulose degradation. Overall, the inoculants were responsible for 20% to 30% of the total observed degradation of the lignocellulose components.

3.2. Influence of the Inoculants on Lignocellulose-Degrading Enzyme Activities

The main secreted ligninolytic enzymes that are responsible for the degradation of lignocellulose are lignin peroxidase (Lip), manganese peroxidase (Mnp), and laccase (Lac) [25]. These enzymes facilitate the oxidation of lignocelluloses, generating free radicals with high oxidation-reduction potential that dismantle aromatic rings and achieve lignocellulose degradation [26]. Their activities were determined in water extracts of the composting material at the end of the experiment. Figure 2A shows that the enzyme activities were significantly higher in the three inoculation treatments compared to CK. The Lip activity was determined as 2568 U/L in F11 and as high as 3793 U/L in FP4. The latter was 120% higher than CK (p < 0.05). The Mnp activity of Q1 and FP4 was identical (4530 U/L), which was 114% higher than CK (p < 0.05). The activity of Lac from Q1 (2562 U/L) was 77% higher than CK (p < 0.05). For most composting materials, the highest activity was seen for Mnp, followed by Lip and then Lac, though this trend was stronger for Q1 and FP4 than for CK, and in the case of F11, Lip activity was more active than the other two enzymes. The Lip activity of Q1 was 33% higher than that of F11, and its Mnp activity was 93% higher. For FP4, these enzymes were 47% and 93% more active than those of F11 (p < 0.05). No significant difference was found in the Lac activity among F11, Q1, and FP4 treatments.

The above results indicated that the inoculation of F11, Q1, and FP4 all increased the activity of the three main lignase enzymes, while Q1 and FP4 inoculation performed better for Lip and Mnp, and Q1 inoculation showed the best performance for all three lignase activities combined.

Xylanase activity was shown to be essential for hemicellulose degradation [18]. The amorphous part of cellulose is easier to degrade due to its loose, microporous structure. Carboxymethyl cellulase (CMCase) mainly acts on the amorphous area inside cellulose, and it has endoglucanase activity, which participates in cellulose degradation [27]. Both xylanase and CMCase activities were determined, but they did not significantly differ among the four treatments (Figure 2B,C).

Lignin is relatively resistant to enzyme hydrolysis owing to its water-insoluble, irregular, and highly branched nature [28]. Nevertheless, the three inoculants facilitated lignocellulose degradation, which could contribute to higher Lip, Mnp, and Lac activities. These secreted enzymes have been intensively studied in white-rot fungi, which can oxidize the lignin polymer [29]. They are also the enzymes by which Actinomycetes inoculants stimulate the degradation of lignin [18]. Zhu and colleagues indicated that the decomposition rate of cellulose, hemicellulose, and lignin was significantly increased due to higher activities of xylanase, Mnp, and Lac [21], but the strains tested here did not result in increased xylanase activity.

No significant difference in the degradation rate of lignin was observed among the three inoculation treatments, which may be caused by the maximum composting temperature (50 °C) reached. The strains tolerate higher temperatures: F11, Q1, and FP4 could resist 55 °C, 60 °C, and 70 °C, respectively. At their permissive temperatures, differences in their lignin degradation rates might be observed, whereas the lower temperature reached here may have weakened potential differences between the strains.

Overall, all three treatments elevated Lip, Mnp, and Lac activities but not CMCase or Xylanase activities. With Lip and Mnp being higher in Q1 and FP4, those two enzymes are responsible for the treatment rates of hemicellulose and cellulose. The contribution of lignocellulosic enzymes following inoculation of the three thermotolerant strains was demonstrated for ligninase, resulting in increased lignin degradation, so our first hypothesis was verified.

3.3. Influence of Thermotolerant Bacteria on Microbial Community Composition and Diversity

During treatment, the inoculated bacteria compete with indigenous microorganisms, which is expected to affect the composition and diversity of the microbial community. To assess these effects, the bacterial and fungal communities following the different treatments were characterized by partial rRNA gene sequencing. From the sequencing data, the Chao and ACE indices were calculated, which increase with community richness. A higher Shannon and lower Simpson index indicate higher community diversity, and these were also determined.

Table 1. Bacterial and fungal diversity indices of different treatments.

	Bacterial Diversity Index				Fungal Diversity Index
Treatment	Chao	Ace	Shannon	Simpson	Chao	Ace	Shannon	Simpson
CK	401.7 a	391.4 a	3.8 a	0.061 a	117.0 ab	123.9 a	0.945 b	0.689 b
F11	373.7 a	364.1 a	3.9 a	0.060 a	139.3 a	125.4 a	1.365 a	0.408 c
Q1	360.9 b	356.6 a	3.5 b	0.096 a	96.7 b	98.0 b	0.666 c	0.798 a
FP4	375.6 ab	362.7 a	3.7 ab	0.079 a	97.7 b	99.5 b	0.589 c	0.819 a

Note: lowercase letters indicate the significance of the difference between different treatments for a given index (p < 0.05).

summarizes the effects of the inoculants on these indices.

Compared to CK, the Chao and Shannon index of bacteria present in the compost following Q1 treatment were significantly lower, by approximately 19%, while those indices had not changed significantly for F11 and FP4 treatment. As to the fungal community, the Chao index had not significantly changed compared to CK (though the Chao of F11 was higher than that of the other two inoculants), but the Shannon index from F11 was higher than CK (45% increase), while that of Q1 and FP4 had decreased by 30% and 38%, respectively. The fungal ACE index from Q1 and FP4 had decreased by approximately 20% and the Simpson index had increased in these two treatments compared to the control, while that of F11 had decreased. Taken together, among the three inoculant treatments, Q1 decreased the bacterial community richness and diversity, F11 increased the fungal community richness and diversity, while both Q1 and FP4 decreased fungal community richness and diversity.

A principal coordinates analysis was performed on the obtained bacterial and fungal communities, with results for all triplicate treatments shown in Figure 3. This demonstrated some variation within the triplicates of each treatment. As a result of the Q1 treatment, the bacterial community (yellow, Figure 3A) was separated from other treatments along both principal component axes. For the fungal community, the Q1 treatment (yellow, Figure 3B) was separated along the PC2 axis and from FP4 for both axes. Overall, strong differences were noted between the PCoA segregation of bacterial and fungal genera as a result of the different treatments.

The sequence data were next used to determine to which OTUs they belonged. The top 10 OTUs representing the highest relative abundance in each sample can be considered dominant [30]. The inoculation of the three Bacillus strains caused changes in bacteria communities at the genus level. Surprisingly, only inoculant F11 resulted in a noticeable higher relative abundance of Bacillus, while no obvious differences were found between CK and Q1 or FP4 (Figure 4A). This would suggest that strain F11 is more suitable for growing in this compost environment than Q1 and FP4.

Members of the genera Nocardiopsis, Brachybacterium, and Halomonas have been proven to be able to produce lignocellulolytic enzymes [31]. Not all of these were highly abundant in the composting materials analyzed here, but noticeable increases in their abundance were seen as a result of treatment (Figure 4B). Nocardiopsis was highly abundant in all samples and increased by 49% in Q1 and by 31% in FP4 compared to control. Compared to CK, the relative abundance (r.a.) of Brachybacterium increased by 59% in F11, by 84% in Q1, and it doubled (100% increase) in FP4. Lastly, the r.a. of Halomonas increased by 23.6% in Q1 compared to CK. Thus, in all 3 treatments, some of these lignocellulase-producing bacteria had increased in abundance, and with inoculum Q1, the relative abundance of three of these four genera had increased. Interestingly, the inoculants produced this effect even though they may not become highly abundant. Although their direct contribution to lignocellulose degradation was not demonstrated and might be minor, their presence induces important shifts in the bacterial community, and in this way, they improved the lignocellulose degradation significantly.

Neither Q1 nor FP4 inoculation increased the r.a. of Bacillus, while F11 did increase the r.a. of Bacillus species. This result is consistent with previous studies, indicating the important ecological role of Bacillus in the composting process. For example, some studies have pointed out that during the composting of citrus peels, microbial inoculation leads to Bacillus species becoming the dominant genera, resulting in a significant reduction in cellulose content during the process. Therefore, the hypothesis that the inoculant would increase the relative abundance of Bacillus was only partially verified [32]. The enhanced enzyme activities and accelerated lignocellulose degradation rates observed with Q1 or FP4 treatments are likely indirect consequences of alterations in the bacterial community structure.

As a side effect, inoculation might decrease the abundance of phytopathogenic bacteria. Decreases in r.a. of bacterial genera were also noted as a result of treatment, for instance for Parapusillimonas and Rhodococcus and the families Xanthomonadaceae, Flavobacteriaceae, and Fodinicurvataceae. The strongest decrease, compared to CK, was observed for Xanthomonadaceae in FP4 (minus 72%). The genus Xanthomonas exhibits a highly phytopathogenic diversity [33]. Rhodococcus decreased strongly in Q1 (minus 41%). Bioremediation using various bacterial strains of the genus Rhodococcus has proved to be a promising option for the clean-up of aromatic compounds and organic nitriles [34]. The presence of plant pathogens can also be affected by bacteria. For instance, colonization of tomato roots by oomycetes was associated with a shift in the microbial community involving a Bacteroidetes to Proteobacteria transition and Flavobacteriaceae as the most abundant family that was affected [35]. The presence of F11, Q1, and especially FP4 decreased the r.a. of these bacteria and may thus decrease the pathogenic potential of the compost.

Sodiomyces was identified as the dominant fungal genus in all treatments, with a relative abundance of approximately 93% in all samples (Supplementary Materials). No obvious differences in the relative abundance of Sodiomyces were detected. This corroborates the relatively small differences noted in the PCoA analysis presented in Figure 3, where the scale of the fungal plot differed by a factor of 5 to 6.6 compared to the bacterial plot.

4. Conclusions

The three Bacillus strains, which had different resistance to high temperatures, improved lignocellulose degradation in composting as a result of increased lignocellulose-degrading enzyme activity. Only the F11 treatment resulted in a higher relative abundance of Bacillus in the compost, but all strains increased Lip, Mnp, and Lac activities to different degrees. Shifts in the bacterial community structure were noted at the bacterial genus level, and inoculation resulted in a higher relative abundance of one or more of the genera Nocardiopsis, Brachybacterium, and Halomonas, which could be the reason for improved lignocellulose degradation, as members of these genera produce more lignocellulose-degrading enzymes. Further research can explore the effects of other potential microbial co-inoculations on lignocellulose degradation under different environmental conditions, such as temperature and humidity. Additionally, a deeper analysis of the interaction mechanisms within microbial communities will help optimize the composting process and improve the quality of organic fertilizers, thereby promoting sustainable agricultural development. Integrating research in these areas will provide new insights into enhancing composting efficiency and resource utilization.