Improved engineered fungal-bacterial commensal consortia simultaneously degrade multiantibiotics and biotransform food waste into lipopeptides.

Resource utilization of food waste is necessary to reduce environmental pollution. However, antibiotics can enter the environment through food waste, resulting in antibiotic residues, which pose potential risks to human health. In this study, commensal artificial consortia were constructed through intercellular adaptation to simultaneously degrade antibiotics and bioconvert food waste into lipopeptides. The biodegradation efficiency of oxytetracycline in the three-strain consortium, which contained lipopeptide-producing Bacillus amyloliquefaciens HM618, high-level proline-producing Corynebacterium glutamate, and laccase-producing Pichia pastoris, was around 100% in the food waste medium at 72 h; this was higher than that in the pure culture of P. pastoris-Lac. Sulfamethoxazole could be removed at 48 h. However, the lipopeptide level in the three-strain consortium was only 77 mg/L. The four-strain consortium containing free fatty acid-producing Yarrowia lipolytica improved the lipopeptide level to around 218 mg/L. The degradation efficiency of oxytetracycline in the four-strain consortium was 100% at 48 h; however, only 56% of the sulfamethoxazole was removed over 96 h. Three five-strain consortia were formed by introducing recombinant manganese peroxidase-producing P. pastoris, recombinant HM618 with high-level amylase, and serine-producing C. glutamicum. In low starch food waste, the highest degradation efficiency of sulfamethoxazole was 71%, while oxytetracycline could be completely removed at 48 h. However, oxytetracycline inhibited starch degradation and lipopeptide production. The high level of starch improved lipopeptide synthesis to 1280 mg/L. The results of this study provide a feasible strategy for the resource utilization of inferior biomass food waste.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

Integrated Biofilm Modification and Transcriptional Analysis for Improving Fengycin Production in Bacillus amyloliquefaciens.

Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41 mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positive regulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172 g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.

Combinatorial metabolic engineering of Bacillus subtilis for de novo production of polymyxin B.

Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into Bacillus subtilis, and sfp from Paenibacillus polymyxa was expressed heterologously, enabling recombinant B. subtilis to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of pmxA, pmxB, and pmxE with PfusA, C2up, and PfusA, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering.

Improving salt-tolerant artificial consortium of Bacillus amyloliquefaciens for bioconverting food waste to lipopeptides.

High-salt content in food waste (FW) affects its resource utilization during biotransformation. In this study, adaptive laboratory evolution (ALE), gene editing, and artificial consortia were performed out to improve the salt-tolerance of Bacillus amyloliquefaciens for producing lipopeptide under FW and seawater. High-salt stress significantly decreased lipopeptide production in the B. amyloliquefaciens HM618 and ALE strains. The total lipopeptide production in the recombinant B. amyloliquefaciens HM-4KSMSO after overexpressing the ion transportor gene ktrA and proline transporter gene opuE and replacing the promoter of gene mrp was 1.34 times higher than that in the strain HM618 in medium containing 30 g/L NaCl. Lipopeptide production under salt-tolerant consortia containing two strains (HM-4KSMSO and Corynebacterium glutamicum) and three-strains (HM-4KSMSO, salt-tolerant C. glutamicum, and Yarrowia lipolytica) was 1.81- and 2.28-fold higher than that under pure culture in a medium containing FW or both FW and seawater, respectively. These findings provide a new strategy for using high-salt FW and seawater to produce value-added chemicals.

Enhancement of polymyxin B1 production by an artificial microbial consortium of Paenibacillus polymyxa and recombinant Corynebacterium glutamicum producing precursor amino acids.

Polymyxin B, produced by Paenibacillus polymyxa, is used as the last line of defense clinically. In this study, exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of polymyxin B analogs of P. polymyxa CJX518-AC (PPAC) from 0.15 g/L and 61.8 % to 0.33 g/L and 79.9 %, respectively. The co-culture of strain PPAC and recombinant Corynebacterium glutamicum-leu01, which produces high levels of threonine, leucine, and isoleucine, increased polymyxin B1 production to 0.64 g/L. When strains PPAC and C. glu-leu01 simultaneously inoculated into an optimized medium with 20 g/L peptone, polymyxin B1 production was increased to 0.97 g/L. Furthermore, the polymyxin B1 production in the co-culture of strains PPAC and C. glu-leu01 increased to 2.21 g/L after optimized inoculation ratios and fermentation medium with 60 g/L peptone. This study provides a new strategy to improve polymyxin B1 production.

An Engineered Microbial Consortium Provides Precursors for Fengycin Production by Bacillus subtilis.

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.

Enhancing fengycin production in the co-culture of Bacillus subtilis and Corynebacterium glutamicum by engineering proline transporter.

Fengycin possesses antifungal activity but has limited application due to its low yields. Amino acid precursors play a crucial role in fengycin synthesis. Herein, the overexpression of alanine, isoleucine, and threonine transporter-related genes in Bacillus subtilis increased fengycin production by 34.06%, 46.66%, and 7.83%, respectively. Particularly, fengycin production in B. subtilis reached 871.86 mg/L with the addition of 8.0 g/L exogenous proline after enhancing the expression of the proline transport-related gene opuE. To overcome the metabolic burden caused by excessive enhancement of gene expression for supplying precursors, B. subtilis and Corynebacterium glutamicum which produced proline, were co-cultured, which further improved fengycin production. Fengycin production in the co-culture of B. subtilis and C. glutamicum in shake flasks reached 1554.74 mg/L after optimizing the inoculation time and ratio. The fengycin level in the fed-batch co-culture was 2309.96 mg/L in a 5.0-L bioreactor. These findings provide a new strategy for improving fengycin production.

Mutualistic microbial community of Bacillus amyloliquefaciens and recombinant Yarrowia lipolytica co-produced lipopeptides and fatty acids from food waste.

Bioconversion is an important method for transforming food waste (FW) into high value-added products, rendering it harmless, and recycling resources. An artificial microbial consortium (AMC) was constructed to produce FW-based lipopeptides in order to investigate the strategy of FW bioconversion into value-added products. Exogenous fatty acids as a precursor significantly improved the lipopeptide production of Bacillus amyloliquefaciens HM618. To enhance fatty acid synthesis and efflux in AMC, the recombinant Yarrowia lipolytica YL21 (strain YL21) was constructed by screening 12 target genes related to fatty acids to replace exogenous fatty acids in order to improve lipopeptide production. The levels of fengycin, surfactin, and iturin A in the AMC of strains HM618 and YL21 reached 76.19, 192.80, and 31.32 mg L-1, increasing 7.24-, 12.13-, and 3.23-fold compared to the results from the pure culture of strain HM618 in flask with Landy medium, respectively. Furthermore, free fatty acids were almost undetectable in the co-culture of strains HM618 and YL21, although its level was around 1.25 g L-1 in the pure culture of strain YL21 with Landy medium. Interestingly, 470.24 mg L-1 of lipopeptides and 18.11 g L-1 of fatty acids were co-produced in this AMC in a bioreactor with FW medium. To our knowledge, it is the first report of FW biotransformation into co-produce of lipopeptides and fatty acids in the AMC of B. amyloliquefaciens and Y. lipolytica. These results provide new insights into the biotransformation potential of FW for value-added co-products by AMC.

Fermentation optimization of surfactin production of Bacillus amyloliquefaciens HM618.

This work isolated a strain named Bacillus amyloliquefaciens HM618 from the soil, which can inhibit the growths of Botrytis cinerea, Rhizoctonia solani, and Escherichia coli DH5α. Based on the results of response surface methodology, the surfactin levels of strain HM618 were elevated from 0.724 to 1.876 g/L and 0.995 to 1.888 g/L under the pure culture with the optimized medium (containing 62.39 g/L sucrose, 15.06 g/L yeast extracts, and 3.27 g/L aspartate) and under the coculture of strains HM618 and Bacillus subtilis 168 with the optimized medium (containing 50.52 g/L sucrose, 19.76 g/L yeast extracts, and 1.02 g/L glutamate), respectively. Additionally, influences of nonconstitutive amino acids involved in the biosynthesis of surfactin were also explored. The highest surfactin level reached 2.04 g/L after adding 3.0 g/L exogenous ornithine. However, the surfactin production of strain HM618 was significantly inhibited after adding the mixtures of nonconstitutive amino acids.

Improved Production of Fengycin in Bacillus subtilis by Integrated Strain Engineering Strategy.

Fengycin is a lipopeptide with broad-spectrum antifungal activity. However, its low yield limits its commercial application. Therefore, we iteratively edited multiple target genes associated with fengycin synthesis by combinatorial metabolic engineering. The ability of Bacillus subtilis 168 to manufacture lipopeptides was restored, and the fengycin titer was 1.81 mg/L. Fengycin production was further increased to 174.63 mg/L after knocking out pathways associated with surfactin and bacillaene synthesis and replacing the native promoter (PppsA) with the Pveg promoter. Subsequently, fengycin levels were elevated to 258.52 mg/L by upregulating the expression of relevant genes involved in the fatty acid pathway. After blocking spore and biofilm formation, fengycin production reached 302.51 mg/L. Finally, fengycin production was increased to approximately 885.37 mg/L after adding threonine in the optimized culture medium, which was 488-fold higher compared with that of the initial strain. Integrated strain engineering provides a strategy to construct a system for improving fengycin production.

Artificial microbial consortium producing oxidases enhanced the biotransformation efficiencies of multi-antibiotics.

Antibiotic mixtures in the environment result in the development of bacterial strains with resistance against multiple antibiotics. Oxidases are versatile that can bio-remove antibiotics. Various laccases (LACs), manganese peroxidases (MNPs), and versatile peroxidase (VP) were reconstructed in Pichia pastoris. For the single antibiotics, over 95.0% sulfamethoxazole within 48 h, tetracycline, oxytetracycline, and norfloxacin within 96 h were bio-removed by recombinant VP with α-signal peptide, respectively. In a mixture of the four antibiotics, 80.2% tetracycline and 95.6% oxytetracycline were bio-removed by recombinant MNP2 with native signal peptide (NSP) within 8 h, whereas < 80.0% sulfamethoxazole was bio-removed within 72 h, indicating that signal peptides significantly impacted removal efficiencies of antibiotic mixtures. Regarding mediators for LACs, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) resulted in better removal efficiencies of multi-antibiotic mixtures than 1-hydroxybenzotriazole or syringaldehyde. Furthermore, artificial microbial consortia (AMC) producing LAC2 and MNP2 with NSP significantly improved bio-removal efficiency of sulfamethoxazole (95.5%) in four-antibiotic mixtures within 48 h. Tetracycline and oxytetracycline were completely bio-removed by AMC within 48 and 72 h, respectively, indicating that AMC accelerated sulfamethoxazole, tetracycline, and oxytetracycline bio-removals. Additionally, transformation pathways of each antibiotic by recombinant oxidases were proposed. Taken together, this work provides a new strategy to simultaneously remove antibiotic mixtures by AMC.

Artificial consortia of Bacillus amyloliquefaciens HM618 and Bacillus subtilis for utilizing food waste to synthetize iturin A.

Food waste is a cheap and abundant organic resource that can be used as a substrate for the production of the broad-spectrum antifungal compound iturin A. To increase the efficiency of food waste biotransformation, different artificial consortia incorporating the iturin A producer Bacillus amyloliquefaciens HM618 together with engineered Bacillus subtilis WB800N producing lipase or amylase were constructed. The results showed that recombinant B. subtilis WB-A13 had the highest amylase activity of 23406.4 U/mL, and that the lipase activity of recombinant B. subtilis WB-L01 was 57.5 U/mL. When strain HM618 was co-cultured with strain WB-A14, the higher yield of iturin A reached to 7.66 mg/L, representing a 32.9% increase compared to the pure culture of strain HM618. In the three-strain consortium comprising strains HM618, WB-L02, and WB-A14 with initial OD600 values of 0.2, 0.15, and 0.15, respectively, the yield of iturin A reached 8.12 mg/L, which was 38.6% higher than the control. Taken together, artificial consortia of B. amyloliquefaciens and recombinant B. subtilis can produce an increased yield of iturin A, which provides a new strategy for the valorization of food waste.

Co-culture of Bacillus amyloliquefaciens and recombinant Pichia pastoris for utilizing kitchen waste to produce fengycins.

Kitchen waste (KW) is a vast potential source of fermentable substrates. To bio-convert the KW into high-value chemicals, we used KW as substrate for the production of fengycin by an artificial consortium containing Bacillus amyloliquefaciens HM618 producing fengycin and the engineering Pichia pastoris producing amylase, glucosidase, or lipases. The maximal amylase activity of the constructed amylase-producing engineering strain (recombinant P. pastoris GS115-amy98) reached 385.4 U‧mL-1. The engineering strain GS115-α-glu53 producing glucosidase reached an enzyme activity titer of 247.3 U‧mL-1, while the lipase activities of the engineering strains GS115-lip2, GS115-α-lip2, and GS115-lip7 were around 90.0 U‧mL-1, with no significant differences among them. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that the components of fengycin synthesized by B. amyloliquefaciens HM618 were complex, including C14-C18 fengycins A, C13-C14 fengycins B, C16-C18 fengycins B, C16 fengycin B2 and some fengycin homologues with unsaturated fatty acid chains. The levels of fengycin were 15.9 mg‧L-1 and 4.6 mg‧L-1 under the co-culture with strain HM618 and the recombinant strains producing amylase and lipase, respectively. The maximal titer of fengycin was 21.2 mg‧L-1 in the artificial consortia consisting of HM618 and the engineering strains producing glucosidase, amylase and lipase. Taken together, these results show that the co-culture of B. amyloliquefaciens HM618 and engineering strains producing amylase and lipase can promote the conversion of KW into fengycin. The work provides a new strategy for boosting the resource utilization of KW.

Improved the lipopeptide production of Bacillus amyloliquefaciens HM618 under co-culture with the recombinant Corynebacterium glutamicum producing high-level proline.

The application of antibacterial lipopeptides is limited by high cost and low yield. Herein, the exogenous L-proline significantly improved lipopeptide production by Bacillus amyloliquefaciens HM618. A recombinant Corynebacterium glutamicum producing high levels of proline using genetically modifying proB and putA was used to establish consortium, to improve lipopeptide production of strain HM618. Compared to a pure culture, the levels of iturin A, fengycin, and surfactin in consortium reached 67.75, 39.32, and 37.25 mg L-1, respectively, an increase of 3.19-, 2.05-, and 1.63-fold over that produced by co-cultures of B. amyloliquefaciens and recombinant C. glutamicum with normal medium. Commercial amylase and recombinant Pichia pastoris with a heterologous amylase gene were used to hydrolyze kitchen waste. A three-strain consortium with recombinant P. pastoris and C. glutamicum increased the lipopeptide production of strain HM618 in medium containing KW. This work provides new strategies to improve lipopeptide production by B. amyloliquefaciens.

Biodegradation of sulfonamide antibiotics through the heterologous expression of laccases from bacteria and investigation of their potential degradation pathways.

In this study, seven laccase genes from different bacteria were linked with the signal peptides PelB, Lpp or Ompa for heterologous expression in E. coli. The recombinant strains were applied for the removal of sulfadiazine (SDZ), sulfamethazine (SMZ), and sulfamethoxazole (SMX). The results obtained for different signal peptides did not provide insights into the removal mechanism. The removal ratios of SDZ, SMZ, and SMX obtained with the recombinant strain 6#P at 60 h were around 92.0%, 89.0%, and 88.0%, respectively. The degradation pathways of sulfonamides have been proposed, including SO2 elimination, hydroxylation, oxidation, pyrimidine ring cleavage, and N-S bond cleavage. Different mediators participate in the degradation of antibiotics through different mechanisms, and different antibiotics have different responses to the same mediator. The addition of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) slightly promoted the removal of sulfonamides by most recombinant strains with different signal peptides, especially for the recombinant strain 2#O. The removal of sulfonamides by 1-hydroxybenzotriazole (HBT) varied with the recombinant strains. Syringaldehyde (SA) had a slight inhibitory effect on the removal of sulfonamides, with the most significant effect on strains 7#L and 7#O.

Potential biotransformation pathways and efficiencies of ciprofloxacin and norfloxacin by an activated sludge consortium.

Fluoroquinolones (FQs), such as ciprofloxacin (CIP) and norfloxacin (NOR), are types of emerging trace pollutants that have attracted great attention. In this study, an activated sludge (AS) consortium with high bio-removal capability to CIP and NOR was obtained by acclimating with CIP and NOR for 10 d. Meanwhile, a CIP- and NOR- transforming bacterial strain (S5), which is highly homologous to the 16S rRNA gene sequence of Enterobacter sp., was isolated from the acclimated AS. The bio-removal efficiency of CIP under the acclimated AS consortium was better than that under the pure culture of Enterobacter sp. S5 (93.1% vs. 89.3%), while the bio-removal efficiency of NOR under the acclimated AS consortium was lower than that under the pure culture of Enterobacter sp. S5 (83.9% vs. 89.8%). The biotransformation and bio-adsorption were two main ways to bio-remove CIP and NOR. However, the CIP and NOR biotransformation efficiencies of the acclimated AS were higher than under the pure culture of Enterobacter sp. S5, while the CIP and NOR adsorption of acclimated AS were lower than that under the pure culture of Enterobacter sp. S5. The N-acetylciprofloxacin and N-acetylnorfloxacin were the main biotransformation products of CIP and NOR. It is possible that acetyltransferase may be involved in the biotransformation process. Whether under the pure culture or AS consortium, the cytotoxicity of CIP and NOR transformation products to gram-negative bacteria was alleviated. Therefore, the acclimated AS and Enterobacter sp. S5 might provide a new strategy for removing contaminants and alleviating of FQs resistance.

Control of the polymyxin analog ratio by domain swapping in the nonribosomal peptide synthetase of Paenibacillus polymyxa.

Polymyxins are used as the last-line therapy against multidrug-resistant bacteria. However, their further clinical development needs to solve problems related to the presence of heterogeneous analogs, but there is still no platform or methods that can regulate the biosynthesis of polymyxin analogs. In this study, we present an approach to swap domains in the polymyxin gene cluster to regulate the production of different analogs. Following adenylation domain swapping, the proportion of polymyxin B1 increased from 41.36 to 52.90%, while that of B1-1 decreased from 18.25 to 3.09%. The ratio of polymyxin B1 and B3 following starter condensation domain swapping changed from 41.36 and 16.99 to 55.03 and 6.39%, respectively. The two domain-swapping strains produced 62.96% of polymyxin B1, 6.70% of B3 and 3.32% of B1-1. This study also revealed the presence of overflow fluxes between acetoin, 2,3-butanediol and polymyxin. To our best knowledge, this is the first report of engineering the polymyxin synthetase gene cluster in situ to regulate the relative proportions of polymyxin analogs. This research paves a way for regulating lipopeptide analogs and will facilitate the development of novel lipopeptide derivatives.

Bio-removal of tetracycline antibiotics under the consortium with probiotics Bacillus clausii T and Bacillus amyloliquefaciens producing biosurfactants.

The contamination of the aquatic environments by tetracycline antibiotics (TCs) is an increasingly pressing issue. Here, we used the addition of exogenous surfactants and in situ biosynthesis of biosurfactants to remove tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and their mixtures using the co-culture of probiotic Bacillus clausii T and Bacillus amyloliquefaciens HM618 producing surfactin. The addition of exogenous biosurfactants to remove TCs was superior to nonionic surfactants. The maximal bio-removal efficiencies for OTC and CTC among mixed antibiotics under the co-culture of B. clausii T and B. amyloliquefaciens HM618 were 76.6% and 88.9%, respectively, which were both better than the efficiency of the pure culture of B. clausii T. TCs were removed mainly through biotransformation rather than absorption and hydrolysis. The removal efficiency was in the order CTC > OTC > TC. The co-culture of B. clausii T and B. amyloliquefaciens HM618 alleviated the cytotoxicity of OTC and CTC. The toxicity of the biotransformation products was lower than that of the parent compounds. Demethylation, hydroxylation, and dehydration are likely the major mechanisms of TC biotransformation. These results illustrate the potential of using surfactants in the bioremediation of tetracycline antibiotics, and provide new avenues for further exploration of the bioremediation of antibiotics pollution.

The biodegradation of cefuroxime, cefotaxime and cefpirome by the synthetic consortium with probiotic Bacillus clausii and investigation of their potential biodegradation pathways.

Cephalosporin residues in the environment are a great concern, but bioremediation options do exist. Bacillus clausii T reached a removal rate of 100% within 8 h when challenged with a mixture of cefuroxime (CFX), cefotaxime (CTX), and cefpirome (CPR). The co-culture of B. clausii T and B. clausii O/C displayed a higher removal efficiency for the mixture of CFX, CTX and CPR than a pure culture of B. clausii O/C. B. clausii T alleviated the biotoxicity of CFX and CPR. What's more, the biotoxicity of for CFX and CPR transformation products released by the co-culture of B. clausii T and B. clausii O/C was lower than that in pure cultures. Real-time PCR was applied to detect the changes in the expression levels of the relevant antibiotic-resistance genes of B. clausii T during CFX and CPR degradation. The results indicated that CFX and CPR enhanced the expression of the ß-lactamase gene bcl1. Hydrolysis, deacetylation and decarboxylation are likely the major mechanisms of CTX biodegradation by B. clausii. These results demonstrate that B. clausii T is a promising strain for the bioremediation of environmental contamination by CFX, CTX, and CPR.