Thermophilic Consortium Research Articles

Thermophilic Hemicellulases Secreted by Microbial Consortia Selected from an Anaerobic Digester.

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia's capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications.

Decolorization and detoxification of Brilliant Crocein GR by a newly enriched thermophilic consortium

The environmental pollution caused by azo dyes at high temperatures has become an urgent problem. However, little attention has been paid to decolorizing azo dyes by thermophilic consortiums. In this study, a thermophilic bacterial consortium (BCGR-T) mainly composed of two genera, namely, Caldibacillus (70.90%) and Aeribacillus (17.63%) was first enriched, which can decolorize Brilliant Crocein GR (BCGR) at high temperatures (50–75 °C), pH values of 6∼8, dye concentrations (100–400 mg/L) and salinities (1–5%, w/v). The enzyme activity results showed that the azoreductase activity was nearly 8.8 times that of the control (p < 0.01), and the intracellular lignin peroxidase was also highly expressed with enzyme activity of 5.64 U (min−1 mg−1 protein) (p < 0.05), indicated that both azoreductase and intracellular lignin peroxidase played an important part in the decolorization process. Furthermore, seven new intermediate metabolic products, including aniline, phthalic acid, 2-carboxy benzaldehyde, phenylacetic acid, benzoic acid, toluene, and 4-methyl-hexanoic acid, were identified. In addition, functional genes related with the azo dye decolorization, such as those encoding the azoreductase, laccase, FMN reductase, NADPH-/NADH-quinone oxidoreductases and NADPH-/NADH dehydrogenases, catechol dioxygenase, homogentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, gentisate 1,2-dioxygenase, azobenzene reductase, naphthalene 1,2-dioxygenase, benzoate/toluate 1,2-dioxygenase, and anthranilate 1,2-dioxygenase and so on were found in the metagenome of the consortium BCGR-T. Finally, a new decolorization pathway of the thermophilic consortium BCGR-T was proposed. In addition, the phototoxicity of BCGR decreased after decolorization. Overall, the thermophilic consortium BCGR-T could be a promising candidate in the treatment of high concentration azo dye wastewater at high temperatures.

Comparative analysis of hydrogen production and bacterial communities in mesophilic and thermophilic consortia using multiple inoculum sources

This study investigates the hydrogen (H2) production performance and bacterial communities in mesophilic (37 °C) and thermophilic (50 °C) H2-producing consortia derived from different inoculum sources and utilizing food waste as a substrate. This study found notable variations in H2 production characteristics among these consortia. Among the mesophilic consortia (MC), the W-MC obtained with wetland (W) as the inoculum source exhibited the highest hydrogen production (3900 mL·L−1 and 117 mL·L−1·h−1), while among the thermophilic consortia (TC), the FP-TC obtained with forest puddle sediment (FP) as the inoculum source showed the highest performance (2112 mL·L−1 and 127 mL·L−1·h−1). This study reveals that the choice of inoculum source plays a crucial role in determining hydrogen production efficiency. Furthermore, the bacterial community analysis demonstrated varying microbial diversity and richness in different inoculum sources. Clostridium, a well-known H2-producing bacterium, was found in both mesophilic and thermophilic consortia and showed a positive correlation with H2 production. Other bacteria, such as Sporanaerobacter, Caproiciproducens, and Caldibacillus, also exhibited significant correlations with H2 production, suggesting their potential roles in the process. The study highlights the complex interactions between bacterial communities and hydrogen production performance, shedding light on the critical factors influencing this renewable energy source. Overall, this study contributes to our understanding of the microbial ecology and the factors affecting hydrogen production in different temperature conditions, which can have practical implications for optimizing biohydrogen production processes using organic waste substrates.

Bioleaching of polymetallic sulphidic mining residues: influence of increasing solid concentration on microbial community dynamics and metal dissolution

Within the European research project NEMO, a bioleaching strategy was developed for efficient metal extraction from bioleach residue currently heap-leached at Sotkamo (Finland) that still contains sulphidic minerals and valuable metals (Ni, Zn, Co, Cu). The strategy of gradually increasing the solid content with 5% steps allowed the adaptation of the consortium up to 20% (w/w) solid content, with efficient metal dissolution and same dominant bacteria. Largest proportions of Sulfobacillusthermosulfidooxidans while Eh increased suggested it to be most involved in iron oxidation. Acidithiobacilluscaldus was rather found when pH stabilized, in line with a production of protons from sulphur oxidation that maintained low pH. ‘Acidithiomicrobium’ P2 was favoured towards the end of the runs and at 20% (w/w) solids possibly due to its tolerance to Ni. The use of gene abundance to evaluate biomass in the pulp provided complementary results to classical cell counts in the liquid phase, and suggested a key role of bacteria associated to mineral particles in iron oxidation. Scaling-up in 21-L stirred-tank reactor at 20% (w/w) solids had no detrimental effect on bioleaching and confirmed metal extraction rates. ‘Acidithiomicrobium’ P2 and Sb. thermosulfidooxidans remained main actors. However, the biological activity was considerably reduced at 30% (w/w) solid concentration, which may be due to a too drastic environmental change for the bacteria to adapt to higher solid concentration. Efficient bioleaching of Sotkamo bioleaching residue at high solid concentration was demonstrated, as well as the robustness of the selected moderately thermophilic consortium, at laboratory and pilot scales.

Decolorization and toxicity evaluation of simulated textile effluent via natural microbial consortia in attached growth reactors

Textile wastewater is heterogeneous in nature with multiple types of dyes and other chemicals. Considering this point, attached growth bioreactors were constructed, using sludge (SD), alkalophilic (AK), and thermophilic (TH) bacterial consortia to develop an effective dye biodegradation system. Simulated dye wastewater, containing a 50 ppm mixture of three reactive azo dyes, viz. Reactive Blue (RB 221), Reactive Yellow (RY 145), and Reactive Red (RR 195) were treated under batch mode operation for up to 192 h. Overall, the color removal efficiency of the reactors varied from 85 to 94 % in 192 h. The thermophilic consortium achieved maximum decolorization (93.5 %), operated at 50 °C, while the alkalophilic consortium attained maximum COD reduction (93.0 %). Nitrites and nitrates were reduced to (14.7 – 79.3) – (84 – 239.4) mg/L, while sulfates ranged from 624.7 – 1003 mg/L. In addition, scanning electron microscopy (SEM) analysis revealed the biofilm community structure, indicating diverse bacterial interactions among each consortium. The biodegradation of dyes was further confirmed through Fourier-transform infrared spectroscopy (FTIR). Phytotoxicity and cytotoxicity analyses revealed that the dye mixture became non-toxic after treatment. This study outlines that natural microbial consortia are efficient dye decolorizers and confers the possibility of using microbial consortia for upscale biological treatment of dyeing effluents.

The advanced performance of microbial consortium for simultaneous utilization of glucose and xylose to produce lactic acid directly from dilute sulfuric acid pretreated corn stover

BackgroundLignocellulosic feedstocks have attracted much attention as a potential carbon source for lactic acid (LA) production because of their ready availability, sustainability, and renewability. However, there are at least two major technical challenges to producing LA from lignocellulose. Inhibitors derived from lignocellulose pretreatment have a negative impact on the growth of cells producing LA. Furthermore, pentose sugars produced from the pretreatment are difficultly utilized by most LA producers, which is known as the carbon catabolite repression (CCR) effect. This complex feedstock can be utilized by a robust microbial consortium with high bioconversion efficiency.ResultsIn this study, a thermophilic consortium DUT50 producing LA was enriched and employed to improve corn stover (CS) utilization. Enterococcus was the dominant family in the consortium DUT50, accounting for 93.66% of the total abundance, with Lactobacillus, Bacillus, Lactococcus, and Trichococcus accounted for the remaining 2.68%. This consortium could be resistant to inhibitors concentration up to 9.74 g/L (2.88 g/L acetic acid, 2.46 g/L furfural, 2.20 g/L 5-HMF, and 2.20 g/L vanillin derived from pretreatment of CS), and simultaneously metabolizes hexose and pentose without CCR effect. Based on the promising consortium features, an efficient process of simultaneous saccharification and co-fermentation (SSCF) was developed to produce LA from acid pretreated corn stover, in which solid–liquid separation and detoxification were avoided. The key influencing factors were investigated and optimized, including dry biomass and cellulase loading, corn steep liquor powder concentration, and the pre-hydrolysis time. The highest LA titer of 71.04 g/L with a yield of 0.49 g/g-CS was achieved at a dry biomass loading of 20% (w/v), which is the highest LA production from non-detoxified acid pretreated corn stover via the SSCF process without wastewater generation reported to date. The simultaneous metabolism of hexose and pentose revealed collaboration between Enterococcus in the consortium, whereas xylose may be efficiently metabolized by Lactobacillus and Bacillus with low abundance via the pentose phosphate pathway.ConclusionsThe experimental results demonstrated the potential advantage of symbiosis in microbial consortia used for LA production from lignocellulosic biomass.

Efficiency of Thermophilic Bacteria in Wastewater Treatment

The present work was carried out to assess the efficiency of thermophilic bacteria in wastewater treatment. A consortium of four thermophilic bacterial strains was used in this study. At 50 °C, the thermophilic consortium showed a very high efficiency in removing total organic carbon (TOC), which reached around 79% in 60 h. Removal of phosphorous was relatively low at 15.4%, while removal of nitrogen was about 83% in 60 h of incubation time. Dissolved oxygen was significantly decreased from 3.43 ppm down to 0.13 ppm in the same period. The consortium of mesophilic bacteria grown at 37 °C showed a substantially different performance from the thermophilic consortium incubated at 50 °C. For example, after 60 h of incubation at 37 °C, only 58.5% of TOC, 13.2% of phosphorous and 75.9% of nitrogen were removed, respectively. Dissolved oxygen was decreased from 3.43 ppm down to 0.28 ppm in the same period. Efficiency of removal rate by thermophilic conditions is higher than the mesophilic conditions. Properties such as fast growth of thermophiles (compared to mesophiles) may explain the exceptional ability of these thermophilic organisms in waste removal.

Effect of seed sludge source and start-up strategy on the performance and microbial communities of thermophilic anaerobic digestion of food waste

The objective of this study was to determine the effect of seed sludge on the start-up of thermophilic anaerobic digestion (AD) and to compare two methods of converting mesophilic to thermophilic consortium. Seed sludge source played an important role in the start-up while one-step temperature increase was the preferred start-up strategy. Biogas yields, pH and VFA levels of step-wise temperature increase reactors were initially comparable to that of the mesophilic control. However, they showed signs of instability and failure after temperature exceeded 50 °C. A great increase in absolute abundance of bacteria but reduced archaea for step-wise temperature increase reactors likely resulted in the over-supply of AD intermediates which were not consumed in time by methanogens, causing reactor souring. An initial drop in biogas and significant VFA accumulation were observed by one-step temperature increase reactors. However, owing to the successful acclimatization of thermophilic bacteria – Thermotogae, Thermoanaerobacterales, and Thermoanaerobacterium, as well as Methanosarcina, the one-step temperature increase reactor recovered from VFA accumulation and poor biogas yield within 10 days. By adopting the one-step temperature increase strategy and a suitable seed sludge, microbial consortia for thermophilic AD was established effectively, which aided the start-up of thermophilic AD of food waste.

Biodegradation of rice straw using thermophilic consortium for methane production by biochemical digestion

Biodegradation of rice straw using thermophilic consortium for methane production by biochemical digestion

Study of an Antarctic thermophilic consortium and its influence on the electrochemical behavior of aluminum alloy 7075-T6

Common alloys used for the manufacture of aircrafts are subject to different forms of environmental deterioration. A major one is corrosion, and there is a strong body of evidence suggesting that environmental microorganisms initiate and accelerate it. The development of an appropriate strategy to reduce this process depends on the knowledge concerning the factors involved in corrosion. In this work, a biofilm forming bacterial consortium was extracted in situ from the corrosion products formed in an aircraft exposed to Antarctic media. Two thermophilic bacteria, an Anoxybacillus and a Staphylococcus strain, were successfully isolated from this consortium. Two extracellular enzymes previously speculated to participate in corrosion, catalase and peroxidase, were detected in the extracellular fraction of the consortium. Additionally, we assessed the individual contribution of those thermophilic microorganisms on the corrosion process of 7075-T6 aluminum alloy, which is widely used in aeronautical industry, through electrochemical methods and surface analysis techniques.

Characterization of the EPS from a thermophilic corrosive consortium

Biofilm forming microorganisms are known to contribute to the corrosion of metallic materials, as they can attach to surfaces and influence the electrochemical behavior. Extracellular polymeric substances (EPS) produced by these microorganisms play a major role in adhesion and resistance of the biofilm, thus also contributing to corrosion. A better understanding of the composition of EPS could help mitigate the impact of bacterial mediated corrosion. Here, a preliminary characterization of the EPS from a thermophilic consortium isolated from a corroded airplane engine is presented. Analysis revealed five different monosaccharides, with predominance of glucose and manose, but also a significant amount of rhamnose. Glycosyl linkage analysis was also performed. On the lipid fraction, three types of fatty acids were found. The predominant protein found by peptide finger printing was S-Layer protein, related to bacterial adhesion. Morphological characterization of the biofilm forming consortium was carried using confocal and scanning electron microscopy.

Cleaner utilization of electroplating sludge by bioleaching with a moderately thermophilic consortium: A pilot study

The semi-pilot scale bioleaching of electroplating sludge by the moderately thermophilic acidophilic consortium was carried out for the first time. During the microbial cultivation, Leptospirillum ferriphilum CS13, Acidithiobacillus caldus S2, and Sulfobacillus acidophilus CS5 could grow rapidly in a 300 L aeration packed reactor, in which the total suspended cell concentration could fluctuate around 3 × 108 cells/mL and the community structure remained relatively stable. During the bioleaching process, the microbial stock solution could effectively leach heavy metals from electroplating sludge in a stirred reactor within a few hours. Meanwhile, the effects of pH, temperature, the quantity of active culture, and liquid-solid ratio on the bioleaching behavior were also investigated. The optimal conditions for electroplating sludge bioleaching were pH 1.5, temperature 45 °C, bacterial liquid ratio 40%, liquid-solid ratio 4:1 L kg−1, and leaching time 5 h. The total removal rate of various heavy metals in electroplating sludge was over 99%. The bioleaching residue was successfully passed the TCLP test, and the total contents of heavy metals in the residue were also well below the regulatory criteria. In addition, the XRD analysis of the bioleaching residue was also confirmed that the moderately thermophilic consortium bioleaching provided a cleaner process than chemical leaching on the removal of the residual fraction metals, which was feasible and attractive for industrial treatment of electroplating sludge.

Effect of the type and concentration of cellulose and temperature on metabolite formation by a fermentative thermophilic consortium

In this study, we hypothesized that anaerobic biodegradation of cellulose is influenced by cellulose type and concentration, temperature, and their interactions. Cellulose biodegradation by an anaerobic consortium was tested in thermophilic batch experiments that combined cellulase action, hydrolysis, and fermentation. Initially, the main constituents in the inocula were Thermoanaerobacter, Clostridium, and Acetivibrio spp. Four types of cellulose and a range of concentrations were used as feedstock with pathways involving hydrolysis and glycolysis to produce H2, CO2, acetate, and ethanol. Long fibrous cellulose, two types of microcrystalline cellulose, and filter paper squares were tested at several concentrations between 2 and 20 g/l as substrates. The yields ranged between 0.1 and 2.9 mmol H2 and 0.7–2.6 mmol ethanol per g cellulose. The rates ranged between 0.01 and 0.2 mmol H2, 0.03–0.2 mmol CO2, and 0.01–0.05 mmol ethanol per g cellulose·h. Statistical analyses indicated that the rates and yields of metabolite production were influenced by two-way interactions between the temperature, type, and concentration of cellulose. The results suggest that two-way interactions between experimental variables may impact the outcomes in cellulose bioconversion studies.

Enrichment of Mesophilic and Thermophilic Mixed Microbial Consortia for Syngas Biomethanation: The Role of Kinetic and Thermodynamic Competition

Mixed culture-based syngas biomethanation is a robust bioconversion process with high versatility in terms of exploitable feedstocks and potential applications, as it could be operated independently, or coupled to anaerobic digestion systems and in-situ biogas upgrading processes. Typically, the syngas biomethanation consists in the stepwise conversion of syngas into methane through a number of catabolic routes, which may vary considerably depending on the operating conditions. In this study, two enrichments were performed at 37 °C and 60 °C to investigate the effect of the incubation temperature on the microbial selection process and the dominant catabolic routes followed. This was carried out through the characterization of the catabolic routes and the microbial composition of the enriched cultures, and a thermodynamic feasibility study on their metabolic networks. The enrichments resulted in two stable microbial consortia with different patterns of activity. The mesophilic enriched consortium presented a more intricate metabolic network composed by four microbial trophic groups, where aceticlastic methanogenesis contributed to 64.9 ± 8.3% of the CH4 production. The metabolic network of the thermophilic enriched consortium was much simpler, consisting in the syntrophic association of carboxydotrophic hydrogenogens and hydrogenotrophic methanogens. This led to significant differences in methane productivity, corresponding to 1.83 ± 0.27 and 33.48 ± 0.90 mmol CH4/g VSS/h for the mesophilic and the thermophilic enriched consortium, respectively, which would potentially make the thermophilic consortium more suited for industrial applications. 16S rRNA gene amplicon analysis indicated the presence of strains with similarity to Acetobacterium sp., Methanospirillum hungateii, Methanospirillum stamsii and Methanothrix sp. at mesophilic conditions, and Thermincola carboxydiphila and Methanothermobacter sp. at thermophilic conditions, implying a role in the conversion of syngas. The thermodynamic feasibility study demonstrated that the microbial selection was not driven solely by kinetic competition, since thermodynamic limitations also played a significant role defining the dominant catabolic routes.

Influence of particle size on copper recovery from sulfide ore by the moderately thermophilic microorganisms

The bioleaching of copper ore by the defined moderately thermophilic consortium containing Leptospirillum ferriphilum and Acidithiobacillus caldus was carried out in the bench-scale column. Bioleaching experiments showed that the leaching rate was 25% higher than that of a conventional leaching and the acid consumption was reduced by 33% at the optimal leaching condition. Meanwhile, the effect of different particle sizes on the rate of the copper leaching was also investigated, and it was shown that the particle size affects the bioleaching rate significantly in the range of 5–25 mm, in which approximately 89.27% of copper was extracted at 5–10 mm, while only 57.08% at 15–25 mm. The results obtained by X-ray diffraction (XRD) analysis of the mineral samples before and after microbial and chemical leaching revealed that the decrease in particle size of the minerals resulted in an enhancement of complex microbial interactions. Especially for the particle size of 5–10 mm, a significant amount of elemental sulfur and jarosite formed on the surface of the mineral, while it was further confirmed that critical microbe-mineral interactions have taken place on the mineral surface. The results indicated that mineral particle size is an integral factor to improve the copper recovery from ore in heap leaching operation. This will provide a reference to the heap construction for the bioleaching.

English

English

Dynamics of a Perturbed Microbial Community during Thermophilic Anaerobic Digestion of Chemically Defined Soluble Organic Compounds.

Knowledge of microbial community dynamics in relation to process perturbations is fundamental to understand and deal with the instability of anaerobic digestion (AD) processes. This study aims to investigate the microbial community structure and function of a thermophilic AD process, fed with a chemically defined substrate, and its association with process performance stability. Next generation amplicon sequencing of 16S ribosomal RNA (rRNA) genes revealed that variations in relative abundances of the predominant bacterial species, Defluviitoga tunisiensis and Anaerobaculum hydrogeniformans, were not linked to the process performance stability, while dynamics of bacterial genera of low abundance, Coprothermobacter and Defluviitoga (other than D. tunisiensis), were associated with microbial community function and process stability. A decrease in the diversity of the archaeal community was observed in conjunction with process recovery and stable performance, implying that the high abundance of specific archaeal group(s) contributed to the stable AD. Dominance of hydrogenotrophic Methanoculleus particularly corresponded to an enhanced microbial acetate and propionate turnover capacity, whereas the prevalence of hydrogenotrophic Methanothermobacter and acetoclastic Methanosaeta was associated with instable AD. Acetate oxidation via syntrophic interactions between Coprothermobacter and Methanoculleus was potentially the main methane-formation pathway during the stable process. We observed that supplementation of Se and W to the medium improved the propionate turnover by the thermophilic consortium. The outcomes of our study provided insights into the community dynamics and trace element requirements in relation to the process performance stability of thermophilic AD.

16s rRNA gene sequencing and radioisotopic analysis reveal the composition of ammonia acclimatized methanogenic consortia

Different mesophilic and thermophilic methanogenic consortia were acclimatised and enriched to extreme total ammonia (9.0 and 5.0 g NH4+-N L−1, respectively) and free ammonia (1.0 and 1.4 g NH3-N L−1, respectively) levels in this study. [2-14C] acetate radioisotopic analyses showed the dominance of aceticlastic methanogenesis in all enriched consortia. According to 16S rRNA gene sequencing result, in mesophilic consortia, methylotrophic Methanomassiliicoccus luminyensis was predominant, followed by aceticlastic Methanosarcina soligelidi. A possible scenario explaining the dominance of M. luminyensis includes the use of methylamine produced by Tissierella spp. and biomass build-up by metabolizing acetate. Nevertheless, further studies are needed to pinpoint the exact metabolic pathway of M. luminyensis. In thermophilic consortia, aceticlastic Methanosarcina thermophila was the sole dominant methanogen. Overall, results derived from this study demonstrated the efficient biomethanation ability of these ammonia-tolerant methanogenic consortia, indicating a potential application of these consortia to solve ammonia toxicity problems in future full-scale reactors.

Single pot bioconversion of prairie cordgrass into biohydrogen by thermophiles

The aim of the present work was to use a thermophilic consortium for H2 production using lignocellulosic biomass in a single pot. The thermophilic consortium, growing at 60 °C utilized both glucose and xylose, making it an ideal source of microbes capable of utilizing and fermenting both hexose and pentose sugars. The optimization of pH, temperature, and substrate concentration increased the H2 production from 1.07 mmol H2/g of prairie cordgrass (PCG) to 2.2 mmol H2/g PCG by using the thermophilic consortium. A sequential cultivation of a thermostable lignocellulolytic enzyme producing strain Geobacillus sp. strain WSUCF1 (aerobic) with the thermophilic consortium (anaerobic) further increased H2 production with PCG 3-fold (3.74 mmol H2/g PCG). A single pot sequential culturing of aerobic and anaerobic microbes can be sustainable and advantageous for industrial scale production of biofuels.

Fermentation performance optimization in an ectopic fermentation system

Ectopic fermentation systems (EFSs) were developed for wastewater treatment. Previous studies have investigated the ability of thermophilic bacteria to improve fermentation performance in EFS. Continuing this research, we evaluated EFS performance using principle component analysis and investigated the addition of different proportions of cow dung. Viable bacteria communities were clustered and identified using BOX-AIR-based repetitive extragenic palindromic-PCR and 16S rDNA analysis. The results revealed optimal conditions for the padding were maize straw inoculated with thermophilic bacteria. Adding 20% cow dung yielded the best pH values (6.94–8.56), higher temperatures, increased wastewater absorption, improved litter quality, and greater microbial quantities. The viable bacteria groups were enriched by the addition of thermophilic consortium, and exogenous strains G21, G14, G4-1, and CR-15 were detected in fermentation process. The proportion of Bacillus species in treatment groups reached 70.37% after fermentation, demonstrating that thermophilic bacteria, especially Bacillus, have an important role in EFS, supporting previous predictions.