Production of polyhydroxyalkanoates (PHAs) by Bacillus megaterium using food waste acidogenic fermentation-derived volatile fatty acids.

High production costs still hamper fast expansion of commercial production of polyhydroxyalkanoates (PHAs). This problem is greatly related to the cultivation medium which accounts for up to 50% of the whole process costs. The aim of this research work was to evaluate the potential of using volatile fatty acids (VFAs), derived from acidogenic fermentation of food waste, as inexpensive carbon sources for the production of PHAs through bacterial cultivation. Bacillus megaterium could assimilate glucose, acetic acid, butyric acid, and caproic acid as single carbon sources in synthetic medium with maximum PHAs production yields of 9-11%, on a cell dry weight basis. Single carbon sources were then replaced by a mixture of synthetic VFAs and by a VFAs-rich stream from the acidogenic fermentation of food waste. After 72 h of cultivation, the VFAs were almost fully consumed by the bacterium in both media and PHAs production yields of 9-10%, on cell dry weight basis, were obtained. The usage of VFAs mixture was found to be beneficial for the bacterial growth that tackled the inhibition of propionic acid, iso-butyric acid, and valeric acid when these volatile fatty acids were used as single carbon sources. The extracted PHAs were revealed to be polyhydroxybutyrate (PHB) by characterization methods of Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The obtained results proved the possibility of using VFAs from acidogenic fermentation of food waste as a cheap substrate to reduce the cost of PHAs production.

Drawing up a collaborative contract: Amino acid cross-feeding between interspecies bacterial pairs.

Synthetic microbial communities have the potential to enable new platforms for bioproduction of biofuels and biopharmaceuticals. However, using engineered communities is often assumed to be difficult because of anticipated challenges in establishing and controlling community composition. Cross-feeding between microbial auxotrophs has the potential to facilitate coculture growth and stability through a mutualistic ecological interaction. We assessed cross-feeding between 13 Escherichia coli amino acid auxotrophs paired with a leucine auxotroph of Bacillus megaterium. We developed a minimal medium capable of supporting the growth of both bacteria and used the media to study coculture growth of the 13 interspecies pairs of auxotrophs in batch and continuous culture, as well as on semi-solid media. In batch culture, 8 of 13 pairs of auxotrophs were observed to grow in coculture. We developed a new metric to quantify the impact of cross-feeding on coculture growth. Six pairs also showed long-term stability in continuous culture, where coculture growth at different dilution rates highlighted differences in cross-feeding amongst the pairs. Finally, we found that cross-feeding-dependent growth on semi-solid media is highly stringent and enables identification of the most efficient pairs. These results demonstrate that cross-feeding is a viable approach for controlling community composition within diverse synthetic communities.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biopolymer by recombinant Bacillus megaterium in fed-batch bioreactors.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated in a wide variety of microorganisms as intracellular carbon and energy storage compounds. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the most valuable biopolymers because of its superior mechanical properties. Here, we developed a bioprocess utilizing recombinant Bacillus megaterium strain for PHBV over-production from glucose, without any precursor addition. PHA production was performed in a controlled bioreactor by batch and fed-batch modes using wild-type B. megaterium and rec-B. megaterium cells overexpressing the native phaC gene. The effect of oxygen transfer rate on biomass formation and PHA accumulation was also investigated, under different dissolved oxygen levels. Structural and thermal properties of PHA were characterized by GC-FID, 1H-NMR, TGA and DSC analyses. Significantly, the copolymer produced from glucose as the carbon source in rec-B. megaterium was composed of 58 mol% of 3-hydroxyvalerate monomers. After 66 h, rec-B. megaterium cells in fed-batch fermentation with a pre-determined growth rate µ0 = 0.1 h-1 produced the highest CDW (7.7 g L-1) and PHA concentration (6.1 g L-1). Moreover, an exponential glucose feeding profile resulted in 2.2-fold increase in PHA yield compared to batch cultivation. Overall, this study paves the way to an enhanced biopolymer production process in B. megaterium cells, where the highest product yield on cell was obtained as YP/X = 0.8 g g-1.

Enhanced 2-keto-L-gulonic acid production by a mixed culture of Ketogulonicigenium vulgare and Bacillus megaterium using three-stage temperature control strategy.

As a key precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG) was mainly produced from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain (Bacillus spp.) with a low conversion rate for decades. The aim of this study was to enhance the 2-KLG production by co-culturing K. vulgare and Bacillus megaterium using three-stage temperature control (TSTC) strategy. By investigating the temperature effect on the 2-KLG fermentation, the optimum temperatures for the growths of K. vulgare and B. megaterium were 32 °C and 29 °C, respectively, while the optimum temperature for 2-KLG production was 35 °C. We developed a TSTC process: the temperature was kept at 32 °C during the first 16 h of fermentation, then decreased to 29 °C for the following 14 h, and maintained at 35 °C to the end of fermentation. By using this new process, the productivity and yield of 2-KLG from L-sorbose were obtained at 2.19 ± 0.19 g/L/h and 92.91 ± 1.02 g/L in 20-L fermentors for 5 batches, respectively, which were 22.35% and 6.02% higher than that of the control treatment (the single temperature of 29 °C). The increased cell density of K. vulgare during the exponential phase and the enhanced SDH activity (increased by 25.18% at 36 h, 17.14% at 44 h) in the production stage might be the reasons for enhanced 2-KLG conversion rate and yield. Our results demonstrated the feasibility of the TSTC strategy for 2-KLG production.

Structural insights into the psychrophilic germinal protease PaGPR and its autoinhibitory loop.

In spore forming microbes, germination protease (GPR) plays a key role in the initiation of the germination process. A critical step during germination is the degradation of small acid-soluble proteins (SASPs), which protect spore DNA from external stresses (UV, heat, low temperature, etc.). Inactive zymogen GPR can be activated by autoprocessing of the N-terminal pro-sequence domain. Activated GPR initiates the degradation of SASPs; however, the detailed mechanisms underlying the activation, catalysis, regulation, and substrate recognition of GPR remain elusive. In this study, we determined the crystal structure of GPR from Paenisporosarcina sp. TG-20 (PaGPR) in its inactive form at a resolution of 2.5 A. Structural analysis showed that the active site of PaGPR is sterically occluded by an inhibitory loop region (residues 202-216). The N-terminal region interacts directly with the self-inhibitory loop region, suggesting that the removal of the N-terminal pro-sequence induces conformational changes, which lead to the release of the self-inhibitory loop region from the active site. In addition, comparative sequence and structural analyses revealed that PaGPR contains two highly conserved Asp residues (D123 and D182) in the active site, similar to the putative aspartic acid protease GPR from Bacillus megaterium. The catalytic domain structure of PaGPR also shares similarities with the sequentially non-homologous proteins HycI and HybD. HycI and HybD are metal-loproteases that also contain two Asp (or Glu) residues in their active site, playing a role in metal binding. In summary, our results provide useful insights into the activation process of PaGPR and its active conformation.

Bioinoculants play a significant role in shaping the rhizospheric microbial community: a field study with Cajanus cajan.

The present study is an attempt to understand the impact of bioinoculants, Azotobacter chroococcum (A), Bacillus megaterium (B), Pseudomonas fluorescens (P), on (a) soil and plant nutrient status, (b) total resident and active bacterial communities, and (c) genes and transcripts involved in nitrogen cycle, during cultivation of Cajanus cajan. In terms of available macro- and micro-nutrients, triple inoculation of the bioinoculants (ABP) competed well with chemical fertilizer (CF). Their 'non-target' effects were assessed in terms of the abundance and activity of the resident bacterial community by employing denaturing gradient gel electrophoresis (DGGE). The resident bacterial community (16S rRNA gene) was stable, while the active fraction (16S rRNA transcripts) was influenced (in terms of abundance) by the treatments. Quantification of the genes and transcripts involved in N cycle by qPCR revealed an increase in the transcripts of nifH in the soil treated with ABP over CF, with an enhancement of 3.36- and 1.57- fold at flowering and maturity stages of plant growth, respectively. The bioinoculants shaped the resident microflora towards a more beneficial community, which helped in increasing soil N turnover and hence, soil fertility as a whole.

HriGFP Novel Flourescent Protein: Expression and Applications.

Biosensors based on microbial cells have been developed to monitor environmental pollutants. These biosensors serve as inexpensive and convenient alternatives to the conventional lab based instrumental analysis of environmental pollutants. Small monomeric naturally occurring fluorescent proteins (fp) can be exploited by converting them as small biosensing devices for biomedical and environmental applications. Moreover, they can withstand exposure to denaturants, high temperature, and a wide pH range variation. The current study employs newly identified novel fluorescent protein HriGFP from Hydnophora rigida to detect environmental contaminants like heavy metals and organo-phosphorous (pesticide) compounds such as methyl parathion. The HriGFP was initially tested or its expression in bacterial systems (Gram positive and Gram negative) and later on for its biosensing capability in E coli (BL21DE3) for detection of heavy metals and methyl parathion was evaluated. Our results indicated the discrete and stable expression of HriGFP and a profound fluorescent quenching were observed in the presence of heavy metals (Hg, Cu, As) and methyl parathion. Structural analysis revealed heavy metal ions binding to HriGFP via amino acid residues. In-silico-analysis further revealed strong interaction via hydrogen bonds between methyl parathion phosphate oxygen atoms and the amino group of Arg119 of HriGFP. This study implies that HriGFP can act as a biosensor for detecting harmful carcinogenic pesticide like methyl parathion in water resources in the vicinity of heavily pesticide impregnated agricultural lands and heavy metal contaminated water bodies around industrial areas.

Synthesis, structural characterization, in vitro DNA binding, and antitumor activity properties of Ru(II) compounds containing 2(2,6-dimethoxypyridine-3-yl)-1H-imidazo(4,5-f)[1, 10]phenanthroline.

The octahedral Ru(II) complexes containing the 2(2,6-dimethoxypyridine-3-yl)-1H-imidazo(4,5-f)[1, 10]phenanthroline ligand of type [Ru(N-N)2(L)]2+, where N-N = phen (1,10-phenanthroline) (1), bpy (2,2'-bipyridine) (2), and dmb (4,4'-dimethyl-2,2'-bipyridine) (3); L(dmpip) = (2(2,6-dimethoxypyridine-3-yl)1Himidazo(4,5-f)[1, 10]phenanthroline), have been synthesized and characterized by UV-visible absorption, molar conductivity, elemental analysis, mass, IR, and NMR spectroscopic techniques. The physicochemical properties of the Ru(II) complexes were determined by UV-Vis absorption spectroscopy. The DNA binding studies have been explored by UV-visible absorption, fluorescence titrations, and viscosity measurements. The supercoiled pBR322 DNA cleavage efficiency of Ru(II) complexes 1-3 was investigated. The antimicrobial activity of Ru(II) complexes was done against Gram-positive and Gram-negative microorganisms. The in vitro anticancer activities of all the complexes were investigated by cell viability assay, apoptosis, cellular uptake, mitochondrial membrane potential detection, and semi-quantitative PCR on HeLa cells. The result indicates that the synthesized Ru(II) complexes probably interact with DNA through an intercalation mode of binding with complex 1 having slightly stronger DNA binding affinity and anticancer activity than 2 and 3.

Microencapsulation of Bacillus megaterium NCT-2 and its effect on remediation of secondary salinization soil.

Aim: To prolong the shelf life of Bacillus megaterium NCT-2 by preparing microcapsules through spray drying, and evaluate their efficiency in secondary salinisation soil remediation.Methods: The wall material and spray drying conditions were optimised. Morphological characteristics of microcapsule were measured, and soil remediation effects were tested under field conditions.Results: A relatively higher survival rate of B. megaterium microcapsule was obtained with 1:1 of chitosan/maltodextrin (w/w) when spray drying was performed at 150.0 °C, with the feed flow rates of 800 mL h-1 and 1000 mL h-1, respectively. The span value of 0.93 ± 0.01 was obtained under above conditions. Microcapsule survival rate was 64.09 ± 0.12% after 6 months of storage. Moreover, microcapsule successfully decreased NO3- and EC value in strongly saline soil by 46.5 ± 1.48% and 45.2 ± 1.51%, respectively.Conclusion: Bacillus megaterium NCT-2 microcapsules have application potential in the remediation of secondary salinisation soil.

Phragmites australis in combination with hydrocarbons degrading bacteria is a suitable option for remediation of diesel-contaminated water in floating wetlands.

The presence of diesel in the water could reduce the growth of plant and thus phytoremediation efficacy. The toxicity of diesel to plant is commonly explained; because of hydrocarbons in diesel accumulate in various parts of plants, where they disrupt the plant cell especially, the epidemis, leaves, stem and roots of the plant. This study investigated the effect of bacterial augmentation in floating treatment wetlands (FTWs) on remediation of diesel oil contaminated water. A helophytic plant, Phragmites australis (P. australis), was vegetated on a floating mat to establish FTWs for the remediation of diesel (1%, w/v) contaminated water. The FTWs was inoculated with three bacterial strains (Acinetobacter sp. BRRH61, Bacillus megaterium RGR14 and Acinetobacter iwoffii AKR1), possessing hydrocarbon degradation and plant growth-enhancing capabilities. It was observed that the FTWs efficiently removed hydrocarbons from water, and bacterial inoculation further enhanced its hydrocarbons degradation efficacy. Diesel contaminated water samples collected after fifteen days of time interval for three months and were analyzed for pollution parameters. The maximum reduction in hydrocarbons (95.8%), chemical oxygen demand (98.6%), biochemical oxygen demand (97.7%), total organic carbon (95.2%), phenol (98.9%) and toxicity was examined when both plant and bacteria were employed in combination. Likewise, an increase in plant growth was seen in the presence of bacteria. The inoculated bacteria showed persistence in the water, root and shoot of P. australis. The study concluded that the augmentation of hydrocarbons degrading bacteria in FTWs is a better option for treatment of diesel polluted water.

Effect of endophytic Bacillus megaterium colonization on structure strengthening, microbial community, chemical composition and stabilization properties of Hybrid Pennisetum.

BACKGROUND: This study was conducted to analyze the effects of endophytic Bacillus megaterium (BM 18-2) colonization on structure strengthening, microbial community, chemical composition and stabilization properties of Hybrid Pennisetum. RESULTS: The BM 18-2 had successfully colonized in the interior tissues in both leaf and stem of Hybrid Pennisetum. During ensiling, the levels of pH, acetic acid (AA), butyric acid (BA), propionic acid (PA), and the population of yeast and aerobic bacteria were significantly (P > 0.05) lower, while lactic acid bacteria (LAB) and lactic acid (LA) were significantly (P < 0.001) higher with the steps forward of ensiling in with BM 18-2 as compared to without BM 18-2 colonized of Hybrid Pennisetum. During the different ensiling days, at days 3, 6, 15, and 30, the genus Brevundimonas, Klebsiella, Lactococcus, Weissella, Enterobacter, Serratia, etc. population were significantly decreased, while genus Pediococcus acidilactici and Lactobacillus plantarum were significantly influenced in treated groups as compared to control. The genus Lactobacillus and Pediococcus were positively correlated with treatment groups. CONCLUSIONS: It is concluded that the endophytic bacteria strain BM 18-2 significantly promoted growth characteristics and biomass yield before ensiling and after ensiling inoculated with or without Lactobacillus plantarum could improve the distinct changes of the undesirable microbial diversity, chemical composition, and stabilization properties in with BM 18-2 as compared to without BM 18-2 colonized Hybrid Pennisetum. © 2019 Society of Chemical Industry.

Synthetic cell-cell communication in a three-species consortium for one-step vitamin C fermentation.

OBJECTIVES: A three-species consortium for one-step fermentation of 2-keto-L-gulonic acid (2-KGA) was constructed to better strengthen the cell-cell communication. And the programmed cell death module based on the LuxI/LuxR quorum-sensing (QS) system was established in Gluconobacter oxydans to reduce the competition that between G. oxydans and Ketogulonicigenium vulgare. RESULTS: By constructing and optimizing the core region of the promoter, which directly regulated the expression of lethal ccdB genes in QS system, IR3C achieved the best lethal effect. The consortium of IR3C- K. vulgare-Bacillus megaterium (abbreviated as 3C) achieved the highest 2-KGA titer (68.80 ± 4.18 g/l), and the molar conversion rate was 80.7% within 36 h in 5 l fermenter. Metabolomic analysis on intracellular small molecules of consortia 3C and 1C showed that most amino acids (such as glycine, leucine, methionine and proline) and TCA cycle intermediates (such as succinic acid, fumaric acid and malic acid) were significantly affected. These results further validated that the programmed cell death module based on the LuxI/LuxR QS system in G. oxydans could also faciliate better growth and higher production of consortium 3C for one-step fermentation. CONCLUSIONS: We successfully constructed a novel three-species consortia for one-step vitamin C fermentation by strengthening the cell-cell communication. This will be very useful for probing the rational design principles of more complex multi-microbial consortia.

Improvement of bio-cementation at low temperature based on Bacillus megaterium.

A low production rate for calcium carbonate with microbial solidification technology at low temperatures often restricts its application. For this reason, adding urea to the medium and the domestication of Bacillus megaterium at low temperature were proposed to produce more calcium carbonate based on an analysis of growth characteristics, urease activity, and the production rates for calcium carbonate under different conditions. Sand solidification tests were conducted to demonstrate improvements caused by the methods. The results showed that the higher the temperature, the faster the growth of Bacillus megaterium and the stronger the urease activity. Growth was fastest and urease activity strongest at a pH of 8. Adding urea to the medium and the domestication of B. megaterium at low temperature can both improve the production rate, effectively increasing calcium carbonate precipitation at low temperature. Combining the two methods resulted in greater improvement of the production rate for calcium carbonate. The two methods were also found to improve the effect of sand solidification. Therefore, our study provides a solid foundation for the actual engineering application of bio-cementation technology at low temperature.

Isolation and Characterization of a New Ferulic-Acid-Biotransforming Bacillus megaterium from Maize Alkaline Wastewater (Nejayote).

Nejayote is an alkaline wastewater generated during the nixtamalization process. Nejayote contains high-value compounds such as ferulic acid (FA), which is widely employed as a substrate for the biotechnological production of flavors and aromas. In the present study, the isolation, identification, and characterization of a native strain of Bacillus megaterium were performed, and its capacity to produce 4-vinylguaiacol (4VG) from ferulic acid was evaluated by employing growing cell and resting cell systems. Growing cells of native B. megaterium biotransformed 6 mM crude FA in nejayote into 2.1 mM 4VG, reaching a productivity of 0.21 mM h-1 4VG, while nejayote enriched with FA at 10, 15, and 25 mM resulted in the formation of 2.4, 3.8, and 6.2 mM 4VG and productivities of 0.24, 0.38, and 0.51 mM h-1 4VG, respectively. In the resting cell system, from 6 and 25 mM pure FA, 3.5 mM 4VG was produced (0.18 mM h-1 4VG), while at 10 and 15 mM FA, 4.6 and 5.1 mM 4VG (average of 0.24 mM h-1 4VG) were obtained, respectively. The native B. megaterium strain, isolated from nejayote, showed great biotechnological potential to produce 4VG from crude FA contained in this wastewater, in which other Bacillus species, such as B. licheniformis and B. cereus, were unable to grow and biotransform FA into 4VG.

Metal removal and morphological changes of B. megaterium in the presence of a spent catalyst.

Spent catalysts represent an environmental concern, mainly due to their elevated metal content. Although conventional treatment methods for spent catalysts are available, they generate large volumes of potentially harmful wastes and gaseous emissions. To overcome the environmental impact, biotechnological approaches are currently being explored and developed. Thus, the current study assayed the capability of Bacillus megaterium strain MNSH1-9K-1 to remove Al, Ni, V and Ti contained in the spent catalyst coded as ECAT-TL-II. To this end, B. megaterium MNSH1-9K-1 growth and metal uptake abilities in the presence of ECAT-TL-II spent catalyst at 15% (wt/vol) pulp density were evaluated in modified Starkey medium at 37 °C and 200 rpm. The results presented here show B. megaterium resistance capability to the high-metal content residue, and its Al, V and Ni removal ability, in 1,059.15 ± 197.28 mg kg-1 of Al, 43.39 ± 24.13 mg kg-1 of V and 0.58 ± 0.00 mg kg-1 of Ni, corresponding to the 0.79%, 1.63% and 0.46% of each metal content, respectively, while no Ti removal was detected. Besides, it was observed that the sporulation process took place in B. megaterium cells in the presence of the spent catalyst. The results shown in this study suggest the potential of the strain MNSH1-9K-1 for the removal of metals contained in high-metal content residues, contributing also to the knowledge of the metal resistance and removal abilities of B. megaterium in the presence of a spent catalyst, and how morphological cell changes may be occurring while metal removal is taking place.

Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding.

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4-108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4-108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4-108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein-protein docking, as well as conversion studies, using the mutants demonstrated that the iron-sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5-fold increase in 15ß-hydroxyprogesterone, the main product of progesterone conversion by CYP106A2.

The cytochrome P450BM-1 of Bacillus megaterium A14K is induced by 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin: Biophysical, molecular and biochemical determinants.

The current study describes biological changes in Bacillus megaterium A14K cells growing in the presence of 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin (TCDD), the most potent congener of dioxins. The results indicate that the metabolizing of 2,3,7,8-TCDD by BmA14K was accompanied with a novel morphological and biophysical profile typified by the growth of single cells with high levels of biosurfactant production, surface hydrophobicity and cell membrane permeability. Moreover, the TCDD-grown bacteria exhibited a specific fatty acid profile characterized by low ratios of branched/straight chain fatty acids (BCFAs/SCFAs) and saturated/unsaturated fatty acids (SFAs/USFAs) with a specific "signature" due to the presence of branched chain unsaturated fatty acids (BCUFAs). This was synchronized with a significant induction of P450BM-1, an unsaturated fatty acid-metabolizing enzyme in B. megaterium. Subsequently, the profile of oxygenated fatty acids in the TCDD-grown bacteria was typified by the presence of 5,6-epoxy derived from unsaturated C15, C16 and C17 fatty acids, that were absent in control bacteria. A net increase was also detected in both hydroxylated and epoxidized fatty acids, especially those derived from C15:0 and C16:1, respectively, suggesting a specific TCDD-induced "signature" of oxygenated fatty acids in BmA14K. Overall, this study sheds light on the use of B. megaterium A14K as a promising bioindicator/biodegrader of dioxins.

Bacillus megaterium adapts to acid stress condition through a network of genes: Insight from a genome-wide transcriptome analysis.

RNA-seq analysis of B. megaterium exposed to pH 7.0 and pH 4.5 showed differential expression of 207 genes related to several processes. Among the 207 genes, 11 genes displayed increased transcription exclusively in pH 4.5. Exposure to pH 4.5 induced the expression of genes related to maintenance of cell integrity, pH homeostasis, alternative energy generation and modification of metabolic processes. Metabolic processes like pentose phosphate pathway, fatty acid biosynthesis, cysteine and methionine metabolism and synthesis of arginine and proline were remodeled during acid stress. Genes associated with oxidative stress and osmotic stress were up-regulated at pH 4.5 indicating a link between acid stress and other stresses. Acid stress also induced expression of genes that encoded general stress-responsive proteins as well as several hypothetical proteins. Our study indicates that a network of genes aid B. megaterium G18 to adapt and survive in acid stress condition.

Engineering Bacillus megaterium Strains To Secrete Cellulases for Synergistic Cellulose Degradation in a Microbial Community.

Recent environmental concerns have intensified the need to develop systems to degrade waste biomass for use as an inexpensive carbon source for microbial chemical production. Current approaches to biomass utilization rely on pretreatment processes that include expensive enzymatic purification steps for the requisite cellulases. We aimed to engineer a synthetic microbial community to synergistically degrade cellulose by compartmentalizing the system with multiple specialized Bacillus megaterium strains. EGI1, an endoglucanase, and Cel9AT, a multimodular cellulase, were targeted for secretion from B. megaterium. A small library of signal peptides (SPs) with five amino acid linkers was selected to tag each cellulase for secretion from B. megaterium. Cellulase activity against amorphous cellulose was confirmed through a series of bioassays, and the most active SP constructs were identified as EGI1 with the LipA SP and Cel9AT with the YngK SP. The activity of the optimized cellulase secretion strains was characterized individually and in tandem to assess synergistic cellulolytic activity. The combination of EGI1 and Cel9AT yielded higher activity than either single cellulase. A coculture of EGI1 and Cel9AT secreting B. megaterium strains demonstrated synergistic behavior with higher activity than either monoculture. This cellulose degradation module can be further integrated with bioproduct synthesis modules to build complex systems for the production of high value molecules.

Bacillus megaterium strains derived from water and soil exhibit differential responses to the herbicide mesotrione.

The intense use of herbicides for weed control in agriculture causes selection pressure on soil microbiota and water ecosystems, possibly resulting in changes to microbial processes, such as biogeochemical cycles. These xenobiotics may increase the production of reactive oxygen species and consequently affect the survival of microorganisms, which need to develop strategies to adapt to these conditions and maintain their ecological functionality. This study analyzed the adaptive responses of bacterial isolates belonging to the same species, originating from two different environments (water and soil), and subjected to selection pressure by herbicides. The effects of herbicide Callisto and its active ingredient, mesotrione, induced different adaptation strategies on the cellular, enzymatic, and structural systems of two Bacillus megaterium isolates obtained from these environments. The lipid saturation patterns observed may have affected membrane permeability in response to this herbicide. Moreover, this may have led to different levels of responses involving superoxide dismutase and catalase activities, and enzyme polymorphisms. Due to these response systems, the strain isolated from water exhibited higher growth rates than did the soil strain, in evaluations made in oligotrophic culture media, which would be more like that found in semi-pristine aquatic environments. The influence of the intracellular oxidizing environments, which changed the mode of degradation of mesotrione in our experimental model and produced different metabolites, can also be observed in soil and water at sites related to agriculture. Since the different metabolites may present different levels of toxicity, we suggest that this fact should be considered in studies on the fate of agrochemicals in different environments.