Accelerated Co-Composting of Textile Waste Using the New Strains and Microbial Consortium: Evaluation of Maturity, Stability and Microbial Activity

Abstract

In the present work, the impact of three new bacterial strains and their consortium on composting was evaluated using textile waste as a main substrate mixed with paper, cardboard and green waste, The effectiveness of these micro-organisms in accelerating organic matter degradation was tested. For bioaugmentation of composting, three concentrations (4%, 6% and 8%) were applied. Among the three strains tested, one strain and the consortium demonstrated high organic matter degradation potential, achieving a total organic carbon concentration between 19–21%, total Kjeldahl nitrogen between 1.29–1.56%, a C/N ratio between 13–16%, and a temperature exceeding 55 °C. In the current study, mature compost was attained in 10 weeks, instead of the 44 weeks required for conventional composting and the 12 weeks achieved with other strains previously used. Identification of the strains by 16S rRNA sequencing revealed that they belonged to Bacillus sp., Paenibacillus sp., and Enterobacter aerogenes, respectively. These strains are recognized for their remarkable potential to breakdown a broad variety of organic matter, including lignocellulosic molecules. Furthermore, incorporation of bacteria into the waste mixture (either separately or as a consortium) extended the thermophilic phase by 2 weeks in this study, especially Bacillus sp., Paenibacillus sp. and consortium, leading to a significant reduction in compost production time. It is noteworthy that the efficacy of these strains was considerably greater compared with the three previous strains (i.e., Streptomyces cellulosae, Achromobacter xylosoxidans and Serratia liquefaciens), which were isolated from compost and used for bioaugmentation in a previous study. Our results demonstrate that bioaugmentation by endogenous microbial strains and/or their consortium significantly accelerates the composting process.

1. Introduction

Throughout the history of mankind, quality of life has continued to develop dramatically, leading to an enormous growth in the amount and quality of waste being disposed of in the absence of appropriate management measures, which potentially threatens human life [1,2]. Composting is widely used as an effective tool for disposing of waste rich in organic matter, in order to manage the tremendous amount of waste [1]. This process involves the catabolism and anabolism of micro-organisms to biodegrade macromolecular organic matter [3]. It is a process inspired by the functioning of the natural ecosystem, where organic matter is transformed from waste into fertilizer for growing plants, thereby minimizing the requirement for chemical fertilizers [4]. Composting can also contribute to reducing environmental pollution by limiting air and groundwater contamination associated with landfill disposal and incineration. However, the nature of the waste used and its degree of biodegradability may significantly affect the quality and length of the composting process [3].

In this regard, several strategies to improve the composting process have been reported, notably the optimization of operational parameters such as pH, temperature, C/N ratio, etc. [5]; nevertheless, these strategies do not necessarily allow the expected performance of the composting system to be achieved, and it is unlikely that they will succeed in rapidly activating the process [4]. For this reason, many authors have opted for microbial inoculation technology to improve the composting process [4,6,7,8,9,10,11]. Microbial inoculants enhance enzyme activity in compost, contribute to the degradation of tough organic matter and produce more precursors for the polymerization of humic substances, thereby shortening the composting time and improving its quality [12,13]. Recently, the field of microbial inoculants has received considerable focus, and there has been a great amount of research into composting processes, involving the incorporation of specific microbial strains and/or a microbial consortium [13,14].

In this respect, bioaugmentation involving microbial strains is an alternative strategy, whereby micro-organisms, specifically bacteria, with their enzymatic machinery, could effectively promote the degradation of recalcitrant molecules and boost humic substances throughout the composting process [4]. A wide range of exogenous microbial genera was studied, such as Thermobifida, Phanerochaeta, Compostibacillus, Micrococcus, etc., as well as the microbial consortium, widely recognized for its capability to break down lignocellulosic feedstocks [1,4,7,8], but rarely are studies devoted to the application of endogenous microbial genera within bioaugmentation. Bioaugmentation using endogenous micro-organisms could be more effective at degrading tough organic matter, as these micro-organisms are already perfectly adapted to the waste used, regardless of its composition, and could even withstand higher levels of toxicity, thereby attracting a great deal of interest. Ultimately, in-depth knowledge of how to use endogenous micro-organisms through the entire composting process is crucial not only to improve the treatment itself, but also to optimize it. In light of the above, this research is part of a series of studies involving on the isolation of strains which degrade recalcitrant compounds, which nowadays represent a crucial challenge affecting composting performance using the bioaugmentation approach. For this reason, the effectiveness of three new endogenous bacterial species (Bacillus sp., Paenibacillus sp., and Enterobacter aerogenes) was evaluated in order to study their ability to improve the quality and duration of textile waste composting using bioaugmentation. An in-depth comparison was also made between these results and those previously published using other endogenous bacteria (i.e., Streptomyces cellulosae, Achromobacter xylosoxidans and Serratia liquefaciens), particularly in terms of maturity (C/N ratio, temperature, total organic content, total Kjeldahl nitrogen, etc.) and shortening of composting processes. The relevance of this study lies precisely in our awareness that micro-organisms could be the only way to improve composting using the bioaugmentation approach in the future.

2. Materials and Methods

2.1. Characterization of Raw Materials

The mix was prepared as previously described [15].

Table 1. Physical–chemical analysis of raw materials [16].

Physical–Chemical Parameters	Textile Waste	Green Waste	Paper and Cardboard Waste	Norm NF U44-051/A2
Moisture, %	51.28 ± 1.03	61.49 ± 1.41	11.28 ± 1.07	40–60
pH	7.4 ± 0.15	6.6 ± 0.53	7.2 ± 0.35	6.5–8.5
Total organic carbon (TOC), %	31.63 ± 1.48	45.67 ± 1.37	59.35 ± 0.90	>20
Total Kjeldahl nitrogen (TKN), %	0.57 ± 0.04	1.23 ± 0.04	1.05 ± 0.05	-
C/N ratio	55.1	37.2	56.7	20–40

Data refer to the mean ± standard deviation calculated on the average of three samples.

describes the composition of the feedstock. For this, a mixture, labeled Mix C, of the three types of wastes was prepared for composting employing the silo technique in a ratio of 40%:30%:30% for textile wastes, green wastes, and paper and cardboard wastes, respectively. A total of 40 kg of wastes was composted at ambient temperature and rotated three times weekly over 44 weeks. Sampling was performed at four cardinal points (north, east, south, and west).

2.2. Bacterial Strain Isolation

Bacterial strains were isolated through a medium derived from Mix C, through the use of 35 g of this mixture in one liter of distilled water, soaked overnight. Subsequently, the filtrate was complemented with 15 g of agar. Phenotypically distinguishable bacterial isolates were screened and further purified by sequential plating until purified isolates were reached [17]. Purified isolates were maintained at 4 °C as stock cultures.

2.3. Bioaugmentation Culture Conditions

For the bioaugmentation test, three bacterial strains were isolated from the compost mixture. Pre-sterilized containers (dimensions of 0.17 × 0.17 m) were inoculated with 1 kg of sterilized residue from Mix C with different inoculum doses, separately as well as with a consortium (4%, 6% and 8%, v/w). During the 12 weeks of processing at ambient temperature, all pots were shaken daily for aeration. To reveal the bioaugmentation efficiency of our isolates, a control assay (T) was performed with identical experimental settings, and excluding inocula.

2.4. Analysis Methods

Total organic content (TOC, %), was assessed using the methods outlined in [18], while the ash content is calculated after calcination in a muffle furnace. Meanwhile, the total Kjeldahl nitrogen content (TKN, %) was established as detailed by the French Association for Standardization [19]. The C/N ratio was therefore obtained from the percentage of total organic carbon (TOC, %) and total Kjeldahl nitrogen (TKN, %). The temperature assessment was measured as outlined by the French Association for Standardization [19]. A conventional serial-dilution procedure was used for microbial analysis. All analyses were assessed in triplicate (at weeks 0, 2, 4, 6, 8, 10 and 12).

2.5. Identification of Micro-Organisms

The Genomic DNA Purification Kit from Thermo Fisher (Waltham, MA, USA) was employed to extract DNA according to the kit protocol. Afterwards, the 16S rRNA gene was then further processed by polymerase chain reaction (PCR) through the bacterial primers FD1 (5′ AGA GTT TGA TCC TGG CTC AG 3′) and RP2 (5′ ACG GCT ACC TTG TTA CGA CTT 3′) in a thermocycler (Verity ABI, Foster City, CA, USA). The ABI 3130XL capillary sequencer was used to sequence the amplified fragments. Sequence alignments were performed with BLASTn 2.16.0+ software and neighbor-joining tree analysis was performed based on the neighbor-joining (NJ) procedure in MEGA_11-0.2. A molecular biology analysis was carried out in the laboratory of the National Center for Scientific and Technical Research (CNRST) at the Functional Genomics Platform in the city of Rabat, Morocco.

2.6. Data Processing and Statistical Analysis

Results are expressed as the average ± standard deviation (SD) of three experimental replicates. Two-way ANOVA analyses were conducted to establish substantial differences amongst organic matter degradation, strains and the consortium, and Tukey’s tests were further employed to establish differences. A p-value of <0.05 was required to be considered statistically representative. XLSTAT 2024.3 was used to carry out these tests.

3. Results

3.1. Changes in Temperature and Bacterial Growth

The temperature changes and the bacterial concentration recorded throughout the composting process using different inoculum concentrations and their consortium are shown in Figure 1a–f. The changes in bacteria throughout the bioaugmentation process are illustrated in Figure 1a–c. It should be noted that for all strains and their consortium, a remarkable increase was recorded. Furthermore, the strongest growth was recorded with S1, S2 and the consortium at a concentration of 8% within 6 weeks, and the weakest with S3. The bacteria-free negative test (T) showed no bacterial development. Microbial growth using the strains isolated in this study was significantly stronger than that achieved previously [16]. The increase in microbial growth in pots inoculated with S2 and the consortium might be ascribed to the greater content of lignocellulosic compounds in the waste used, as well as to the ability of this strain to assimilate recalcitrant molecules through their enzymatic system [20]. The lower microbial growth recorded with S3 could be attributed to the fact that these strains require a longer time to produce the necessary enzymes to degrade recalcitrant molecules [16].

Temperature provides information about the rate of organic matter decomposition, which is directly correlated with the microbial metabolic activity that occurs through the composting process. In this study, the pots were maintained at room temperature to ensure that the increase in temperature was due to the strain’s metabolism, and therefore to the strain’s efficiency, and not to the artificial heating. In this regard, Figure 1d–f reveals a relatively low conversion of microbial energy in S3, compared with S1, S2 and the consortium, which is confirmed by the temperature value recorded throughout this study. Indeed, pots with a high concentration of inoculum (8%) reach higher temperatures earlier (from the 4th week) than those with a low concentration of inoculum (4%), as well as ambient temperature (AT). The highest temperature was recorded with S2 (57.86 °C) and the consortium (59.12 °C), both at a concentration of 8% within week 8, and the lowest was achieved with S3 (39.22 °C). These results are considerably more impressive than those previously reported with three other strains (Streptomyces cellulosae, 33.26 °C; Achromobacter xylosoxidans, 40.23 °C; and Serratia liquefaciens, 42.56 °C; and their consortium, 54.17 °C) employed in previous studies [16].

Table 2. Changes in TOC, TKC, C/N and temperature using difference strains based on previous study [16], for comparison with current study.

Strains Used		TOC, %	TKN, %	C/N	Temperature, °C
Streptomyces cellulosae	Final values	24.60	1.23	20.00	33.26
Achromobacter xylosoxidans	Final values	23.45	1.18	19.87	40.23
Serratia liquefaciens	Final values	22.20	1.15	19.30	42.56
Consortium	Final values	20.30	1.52	13.36	54.17

proves that the strains used in the current studies are considerably more effective at degrading recalcitrant compounds than those deployed previously. These results are even significantly greater than those obtained by Wang et al. [4], who worked with several exogenous strains, including Bacillus species, but used thermostats to maintain the temperature above 50 °C, and Hemati et al. [21], who worked with several exogenous species from Paenibacillus, confirming the effectiveness of the endogenous strains employed in this study in degrading organic matter. It is noteworthy that the thermophilic phase is crucial for preventing pathogenic micro-organisms that are dangerous to humans and the environment [21]. In this regard, the longer the thermophilisation process (in our case, around 2 weeks), the greater the quality of the compost, as it is rendered increasingly sterile. Throughout this phase, thermophilic micro-organisms with strong lignocellulolytic ability can significantly accelerate the process of composting, which in itself may boost the rate of degradation of recalcitrant matter [7,16,21], which is the case in the current study. Ultimately, the highest temperature achieved with both the S2 strain and the consortium demonstrates that the final compost produced using this strain can be used safely as a soil amendment without any adverse effects on either plant or human health [4]. After being maintained at over 55 °C for 2 weeks, the temperature plunged to around 35 °C, reflecting a cooling and therefore maturation phase. In the group of bioaugmentation pots, the microbial consortium and the S2 strain were predominantly more efficient, both in improving composting temperature and quality, compared with the normal treatment without bioaugmentation and even with other bacterial strains previously used [16,22]. Overall, endogenous bacterial bioaugmentation boosted the compost’s quality by raising the temperature throughout the treatment process and thereby increasing the thermophilic phase period, which in turn shortened the composting time.

3.2. Variations in TOC, Ash, TKN and C/N Ratio During Bioaugmentation

Regarding total organic carbon (TOC), all pots displayed a downward trend attributable to the mineralization process, which was also confirmed by the increase in ash levels (a good indicator of organic matter mineralization) (Figure 2a–f). This decrease was more pronounced in the early stages with S1, S2 and the consortium at 8% inoculum concentration compared with the S3 strain. The reduction in TOC and ash is directly linked to the possibility that the significant amounts of lignocellulosic compounds in the mixture used have been biodegraded and/or assimilated by the inoculated strains and their consortium, given that the TOC in the negative test remains unchanged throughout the composting process. This proves the effectiveness of the strains used, especially S2 [7]. Generally, the rate of TOC degradation and ash in pots inoculated with S2 and the consortium at a concentration of 8% was higher than that in pots inoculated with S1 and S3 at different concentrations (4%, 6% and 8%). These results are also much better than those recorded previously [16], as well as those recorded by other studies using different exogenous strains and different lignocellulosic wastes [4,7,21,23,24,25], indicating that bioaugmentation using S2 and the consortium boosted the mineralization and subsequent humification of biomass.

As shown in Figure 3a–c, the TKN content increased over the treatment period. It should be noted that even for the nitrogen content, the TKN in the pots inoculated with S2 and the consortium at a concentration of 8% was significantly increased compared to those inoculated with S1 and S3 at different concentrations (4%, 6% and 8%). In this respect, the highest value was recorded with S2 (1.5%) and the consortium (1.56%) at a concentration of 8%, and the lowest value at the same concentration was recorded with S3 (1.18%). These results are also considerably higher than those achieved previously [16]. Nevertheless, the TKN level remained unchanged over time in the negative control (T). The increase in TKN throughout the treatment might be explained by the breakdown of nitrogen-rich materials into ammonia compounds by the strains used [26]. The C/N ratio is a significant index of the availability of nutrients for micro-organisms [7]. Figure 3d–f displays the trend in the C/N ratio throughout the composting process using several strains and their consortium. It should be noted that throughout the treatment, the C/N of all the pots displayed a downward tendency owing to the mineralization of the organic matter, itself attributable to the activity of the strains used. In fact, pots with a high concentration of inoculum (8%) reach maturity phase earlier (from the 6th week) than those with a low concentration of inoculum (4%) (from the 10th week). For the negative test (without inoculum), no change was recorded during the composting process, which is expected since the pots are sterilized and there is therefore no microbial activity, proving that the degradation was accomplished by the inoculated strains. Towards the end of the treatment, the C/N achieved with S1, S2 and the consortium dropped below 20 at a concentration of 8%, except for S3 where the C/N was still above 20 even at a higher inoculum concentration (Figure 3d–f). In this regard, microbial activity had a considerable impact on the C/N ratio, and as this activity increased (Figure 1d–f), the level of carbon and non-nitrogenous compounds like carbohydrates dropped as the micro-organisms assimilated these molecules [21,25,26]. Ultimately, the results are considerably more impressive than those achieved in the previous study [16].

3.3. Correlation Between Bacteria, Consortium and Physical-Chemical Parameters

The relationship between bacteria and physical–chemical data was examined by principal component analysis and the Pearson correlation heatmap (Figure 4,

Table 3. Pearson correlation heatmap of the relationship between physical–chemical variables, bacteria as well as the consortium.

	TOC	TKN	C/N	Temperature	Bacillus sp.	Paenibacillus sp.	Enterobacter aerogenes	Consortium
TOC	1
TKN	−0.913	1
C/N	0.888	−0.989	1
Temperature	−0.554	0.607	−0.667	1
Bacillus sp.	−0.606	0.606	−0.616	0.726	1
Paenibacillus sp.	−0.660	0.596	−0.585	0.755	0.949	1
Enterobacter aerogenes	−0.799	0.719	−0.691	0.764	0.922	0.925	1
Consortium	−0.779	0.717	−0.727	0.862	0.932	0.964	0.920	1

Blue color depicts negative correlation and red color positive correlation.

). The results of the PCA between physical–chemical parameters and microbial strains used in bioaugmentation show that the first component (PC 1) represents 81.57%, whereas the second component (PC 2) represents 10.34% of the variability between physical–chemical parameter profiles and microbial strains. It is noteworthy that the temperature, ash and TKN were strongly positively correlated with Paenibacillus sp. and the consortium, and they were significantly inversely related to TOC and C/N (p < 0.05), pointing to a strong impact of microbial strains on organic matter decomposition and compost maturity. These results were further corroborated by the Pearson correlation heat map presented in Table 1, which proves the strong correlation between Paenibacillus sp., the consortium and physical–chemical parameters.

3.4. Identification of Bacteria

FASTA formats were examined with the BLASTn software database. Scores, percent identity, and query coverage were used to identify the isolated strain, requiring greater than 96% to define these strains as: Bacillus sp., Paenibacillus sp., and Enterobacter aerogenes for S1, S2, and S3, respectively (homology exceeding 97%). The sequence of these strains was registered with GenBank under accession numbers JX317684, KP189376 and JQ682634, respectively, for Bacillus sp., Paenibacillus sp. and Enterobacter aerogenes. Three neighbor-joining trees (phylogenetic trees) of these strains were constructed by comparing the 16S rRNA gene homologies with existing data (Figure 5a–c). These phylogenetic trees revealed that S1, S2, and S3 were strongly associated with Bacillus sp., Paenibacillus sp. and Enterobacter aerogenes, respectively.

In this respect, it has been proven that species belonging to Bacillus sp. are able to use a broad variety of organic matter, in particular resistant substances such as hemicellulose and lignocellulosic compounds, as a valuable carbon and energy supply [27], which explains the improved results achieved in this study. Several authors identify Bacillus as a pioneer in boosting the decomposition of lignocellulosic feedstock [27,28]. Its ability to break down these recalcitrant molecules is mainly due to its enzymatic mechanisms, such as cellulase or other mechanisms [27,29]. Furthermore, Wang et al. [30] described how the addition of Bacillus to the bioaugmentation of a mixture of wheat and cattle manure during composting improved the breakdown of lignocellulose and the fertility of the compost; they revealed that the Bacillus used the lignocellulose for its own proliferation, while simultaneously generating monomers, such as amino acids, and reducing sugars. These molecules could be used as precursors and could be polymerized into humic substances during the humification process. Recently, Zhao et al. [27] demonstrated that Bacillus had a considerable impact on the acceleration of humification. Regarding Paenibacillus, extensive research has highlighted its outstanding potential in the decomposition of lignocellulosic compounds [23,31,32,33,34]. Penibacillus is an ancestral member of the Bacillus genus, known for production of a variety of enzymes, especially amylases, oxygenases, cellulases, hemicellulases, dehydrogenases, lipases, lignin-modifying enzymes, pectinases, etc. [31,35]. Furthermore, Plakys et al. [36], have identified several enzymes which hydrolyse polymers such as cellulose, hemicellulose and/or lignin. Further studies have revealed that this ability may be predominantly due to the presence of specific enzymes directly linked in the depolymerisation of these components, and these findings have been confirmed by NGS [22], which explains the high degradation rate by Paenibacillus sp. In fact, several researchers used Paenibacillus sp. for bioremediation [31,33,37]. In this regard, Grady et al. [35] highlighted the contribution of Paenibacillus species towards the elimination or decomposition of the environmental pollutants, either by bioflocculation or the enzymatic activities. They explain that Paenibacillus can generate a variety of enzymes that metabolise aromatic and aliphatic organic contaminants, including the dehydrogenases, oxygenases, and ligninolytic enzymes to degrade these contaminants. Another study has demonstrated the effectiveness of Paenibacillus strains in degrading textile dyes such as polyvinyl alcohol (PVA), a covering for textile and paper materials [37]. This confirms the high performance of Paenibacillus in the decomposition of organic matter throughout this study, which is even more impressive than that previously achieved with the other endogenous strains [16]. Regarding Enterobacter aerogenes, Sharma and Melkania [38] revealed that these species could use lignocellulosic by-products, particularly glucose, for their metabolism, and were unable to use the recalcitrant molecules; this is due to their inability to release the enzyme required to decompose these molecules [39], which therefore explains the low degradation rate results obtained using that strain.

Ultimately, the use of a consortium of isolates achieved a significantly more pronounced, precise and quicker degradation of organic matter than when the isolates were used separately, potentially due to the capacity of the endogenous bacteria involved in the consortium to co-exist with each other. The consortium of these three bacterial strains could apparently promote and boost the degradation of organic matter using different pathways and mechanisms [16,39]. Several studies have shown that synergistic cooperation within a bacterial consortium can significantly accelerate the transformation of organic matter, particularly recalcitrant substances, which explains the results achieved in this study [8,10,39]. The consortium employed proved significantly (p < 0.05) effective in improving composting process parameters, including temperature, TOC, ash, TKN and C/N, rapidly converting organic matter into compost in 10 weeks, instead of 12 weeks using other sets of strains (Streptomyces cellulosae, Achromobacter xylosoxidans, and Serratia liquefaciens), and 44 weeks in the case of conventional composting without bioaugmentation. In this respect, the high rate of recalcitrant molecule degradation within the consortium could be attributed to the fact that these strains work together to degrade organic matter. For this reason, Bacillus and Paenibacillus, with their enzymatic systems, could act as degraders of lignocellulosic compounds, while Enterobacter aerogenes could use the by-products as a source of carbon and energy.

4. Conclusions

Composting could be efficiently boosted by inoculation with endogenous strains, thereby overcoming the challenges linked to temperature rise, humification and maturation. In this regard, Bacillus sp., Paenibacillus sp. and Enterobacter aerogenes selected for bioaugmentation of textile waste have considerably improved the consistency of the compost produced relative to the conventional treatment without bioaugmentation, and relative to Streptomyces cellulosae, Achromobacter xylosoxidans and Serratia liquefaciens, previously selected for bioaugmentation under the same conditions and with the identical mixture. Within the bacteria tested, Paenibacillus sp. and the bacterial consortium at a concentration of 8% inoculum provided the strongest enhancement in compost consistency and accelerated the composting process. The incorporation of Paenibacillus sp. and the bacterial consortium into the mixture dramatically boosted and lengthened the thermophilic phase. Consequently, the degradation of organic matter, particularly recalcitrant molecules, was considerably improved, and the composting process was shortened, from 44 weeks for the non-bioaugmented compost and 12 weeks for the bioaugmented compost using other strains to 10 weeks in this study. Ultimately, it can be deduced from these results that bioaugmentation with Paenibacillus sp. and the bacterial consortium improved and shortened the composting process, thereby supplying a basis for further practical deployment of accelerated composting technology using different feedstocks. Furthermore, the newly proposed consortium may be used to provide a forward-looking ecological outlook for the durable handling of solid residues.