Treating waste with waste: Lignin acting as both an effective bactericide and passivator to prevent acid mine drainage formation at the source.

Acid mine drainage (AMD) induced by pyrite oxidation is a notorious and serious environmental problem, but the management of AMD in an economical and environmentally friendly way remains challenging. Here, lignin, a natural polymer and abundant waste, was employed as both a bactericide and passivator to prevent AMD formation. The addition of lignin to a mimic AMD formation system inoculated with Acidithiobacillus ferrooxidans at a lignin-to-pyrite weight ratio of 2.5: 10 reduced the combined abiotic and biotic oxidation of pyrite by 68.4 % (based on released SO42-). Morphological characterization of Acidithiobacillus ferrooxidans revealed that lignin could act on the cell surface and impair the cell integrity, disrupting its normal growth and preventing biotic oxidation of pyrite accordingly. Moreover, lignin can be used alone as a passivator to form a coating on the pyrite surface, reducing abiotic oxidation by 71.7 % (based on released SO42-). Through multiple technique analysis, it was proposed that the functional groups on lignin may coordinate with iron ions on pyrite, promoting its deposition on the surface. In addition, the inherent antioxidant activity of lignin may also be actively involved in the abatement of pyrite oxidation via the reduction of iron. Overall, this study offered a "treating waste with waste" strategy for preventing AMD formation at the source and opened a new avenue for the management of AMD.

Integration of lactic acid biorefinery with treatment of red mud from alumina refinery: win-win paradigm for waste valorization.

The cost of detoxification and neutralization poses certain challenges to the development of an economically viable lactic acid biorefinery with lignocellulosic biomass as feedstock. Herein, red mud, an alkaline waste, was explored as both a detoxifying agent and a neutralizer. Red mud treatment of lignocellulosic hydrolysate effectively removed the inhibitors generated in dilute acid pretreatment, improving the lactic acid productivity from 1.0 g/L·h-1 to 1.9 g/L·h-1 in later fermentation. In addition, red mud could replace CaCO3 as a neutralizer in lactic acid fermentation, which in turn enabled simultaneous bioleaching of valuable metals (Sc, Y, Nd, and Al) from red mud. The neutralization of alkali in red mud by acids retained in lignocellulosic hydrolysate and lactic acid produced from fermentation led to effective dealkalization, rendering a maximum alkali removal efficiency of 92.2 %. Overall, this study offered a win-win strategy for the valorization of both lignocellulosic biomass and red mud.

Discrepant assembly processes of prokaryotic communities between the abyssal and hadal sediments in Yap Trench.

Abyssal and hadal sediments represent two of the most type ecosystems on Earth and have the potential interactions with geochemistry. However, little is known about the prokaryotic community assembly and the response of prokaryotic communities to metal(loid)s in trench sediments due to the lack of adequate and appropriate samples. In this study, a systematic investigation combined the assembly mechanisms and co-occurrence patterns of prokaryotic communities between the hadal and abyssal sediments across the Yap Trench. The results revealed that the hadal prokaryotes had less species diversity, but more abundant function than the abyssal prokaryotes. The prokaryotic communities in the abyssal sediments had more core taxa than the hadal sediments. Twenty-one biomarkers mostly affiliated with Nitrosopumilaceae were detected using Random-Forests machine learning algorithm. Furthermore, stochasticity was dominant in the prokaryotic community assembly processes of the Yap Trench sediments. Meanwhile, homogeneous selection (32.6%-52.9%) belonging to deterministic processes governed the prokaryotic community assembly in hadal sediments with increasing of sediment depth. In addition to total nitrogen and total organic carbon, more metal(loid)s were significantly correlated with the prokaryotic community in the hadal sediments than that in the abyssal sediments. The hadal prokaryotic communities was most positively related to bismuth (r = 0.31, p < 0.01), followed by calcium, chromium, cerium, potassium, plumbum, scandium, titanium, and vanadium. Finally, co-occurrence networks revealed two potential dominant prokaryotic modules in Yap Trench sediments covaried across oceanographic zonation. By contrast, the hadal network had relatively more complexity, more bacterial taxa, and more associations among prokaryotic taxa, relative to the abyssal network. This study reveals potentially metal variables and community assembly mechanisms of the prokaryotic community in abyssal and hadal sediments and provides a better understanding on the prokaryotic diversity and ecology in trench sediment ecosystems.

Depth significantly affects plastisphere microbial evenness, assembly and co-occurrence pattern but not richness and composition.

Microplastics have become one of the hot concerns of global marine pollution. In recent years, diversity and abiotic influence factors of plastisphere microbial communities were well documented, but our knowledge of their assembly mechanisms and co-occurrence patterns remains unclear, especially the effects of depth on them. Here, we collected microorganisms on microplastics to investigate how ocean depth affects on microbial diversity, community composition, assembly processes and co-occurrence patterns. Our results indicated that there were similar microbial richness and community compositions but microbial evenness and unique microbes were obviously different in different ocean layers. Our findings also demonstrated that deterministic processes played dominant roles in the assembly of the mesopelagic plastisphere microbial communities, while the bathypelagic microbial community assembly was mainly shaped by stochastic processes. In addition, the co-occurrence networks suggested that the relationships between microorganisms in the mesopelagic layer were more complex and stable than those in the bathypelagic layer. Simultaneously, we also found that Proteobacteria and Actinobacteriota were the most abundant keystones which played important roles in microbial co-occurrence networks at both layers. This study enhanced our understanding of microbial diversity, assembly mechanism, and co-occurrence pattern on plastisphere surfaces, and provided useful insights into microorganisms capable of degrading plastics and microbial remediation.

Depth-Dependent Distribution of Prokaryotes in Sediments of the Manganese Crust on Nazimov Guyots of the Magellan Seamounts.

Deep ocean polymetallic nodules, rich in cobalt, nickel, and titanium which are commonly used in high-technology and biotechnology applications, are being eyed for green energy transition through deep-sea mining operations. Prokaryotic communities underneath polymetallic nodules could participate in deep-sea biogeochemical cycling, however, are not fully described. To address this gap, we collected sediment cores from Nazimov guyots, where polymetallic nodules exist, to explore the diversity and vertical distribution of prokaryotic communities. Our 16S rRNA amplicon sequencing data, quantitative PCR results, and phylogenetic beta diversity indices showed that prokaryotic diversity in the surficial layers (0-8 cm) was > 4-fold higher compared to deeper horizons (8-26 cm), while heterotrophs dominated in all sediment horizons. Proteobacteria was the most abundant taxon (32-82%) across all sediment depths, followed by Thaumarchaeota (4-37%), Firmicutes (2-18%), and Planctomycetes (1-6%). Depth was the key factor controlling prokaryotic distribution, while heavy metals (e.g., iron, copper, nickel, cobalt, zinc) can also influence significantly the downcore distribution of prokaryotic communities. Analyses of phylogenetic diversity showed that deterministic processes governing prokaryotic assembly in surficial layers, contrasting with stochastic influences in deep layers. This was further supported from the detection of a more complex prokaryotic co-occurrence network in the surficial layer which suggested more diverse prokaryotic communities existed in the surface vs. deeper sediments. This study expands current knowledge on the vertical distribution of benthic prokaryotic diversity in deep sea settings underneath polymetallic nodules, and the results reported might set a baseline for future mining decisions.

Regulation on Pathway Metabolic Fluxes to Enhance Colanic Acid Production in Escherichia coli.

Colanic acid (CA) is a natural polysaccharide macromolecule with rich and unique biological properties and is a promising candidate for use in food and cosmetics. To date, the efficient biosynthesis of CA and the influence of product accumulation on the strains used have yet to be precisely investigated. Herein, bottlenecks in the CA metabolic pathway were untangled by finely regulating the expression of manA, cpsG, fcl, and rcsA. Engineered strains produced CA at >1 g/L in shake flasks without dependence on cold temperatures, and it was verified in a 1 L bioreactor with a titer up to 18.64 g/L within 24 h. The accumulation of CA caused a decrease in the saturated fatty acid content (represented by C16:0 and C18:0) in the cell membrane. This study demonstrated pathway engineering for efficient CA production in cell factories and provided insights into the barriers and solutions faced in the biosynthesis of natural products.

Enhanced Enzymatic Performance of ß-Mannanase Immobilized on Calcium Alginate Beads for the Generation of Mannan Oligosaccharides.

Mannan oligosaccharides (MOSs) are excellent prebiotics that are usually obtained via the enzymatic hydrolysis of mannan. In order to reduce the cost of preparing MOSs, immobilized enzymes that demonstrate good performance, require simple preparation, and are safe, inexpensive, and reusable must be developed urgently. In this study, ß-mannanase was immobilized on calcium alginate (CaAlg). Under the optimal conditions of 320 U enzyme addition, 1.6% sodium alginate, 2% CaCl2, and 1 h of immobilization time, the immobilization yield reached 68.3%. The optimum temperature and pH for the immobilized ß-mannanase (Man-CaAlg) were 75 °C and 6.0, respectively. The Man-CaAlg exhibited better thermal stability, a high degree of pH stability, and less substrate affinity than free ß-mannanase. The Man-CaAlg could be reused eight times and retained 70.34% of its activity; additionally, the Man-CaAlg showed 58.17% activity after 30 days of storage. A total of 7.94 mg/mL of MOSs, with 4.94 mg/mL of mannobiose and 3.00 mg/mL of mannotriose, were generated in the oligosaccharide production assay. It is believed that this convenient and safe strategy has great potential in the important field of the use of immobilized ß-mannanase for the production of mannan oligosaccharides.

Distinct assembly processes and environmental adaptation of abundant and rare archaea in Arctic marine sediments.

Revealing the ecological processes and environmental adaptation of abundant and rare archaea is a central, but poorly understood, topic in ecology. Here, abundant and rare archaeal diversity, community assembly processes and co-occurrence patterns were comparatively analyzed in Arctic marine sediments. Our findings revealed that the rare taxa exhibited significantly higher diversity compared to the abundant taxa. Additionally, the abundant taxa displayed stronger environmental adaptation than the rare taxa. The co-occurrence network analysis demonstrated that the rare taxa developed more interspecies interactions and modules in response to environmental disturbance. Furthermore, the community assembly of abundant and rare taxa in sediments was primarily controlled by stochastic and deterministic processes, respectively. These findings provide valuable insights into the archaeal community assembly processes and significantly contribute to a deeper understanding of the environmental adaptability of abundant and rare taxa in Arctic marine sediments.

Kinetic process of the biosorption of Cu(II), Ni(II) and Cr(VI) by waste Pichia pastoris cells.

Waste biomass of Pichia pastoris (P.pastoris) cells from the fermentation industry is an environmentally friendly biosorption material. The present study aimed to explore the biosorption behaviour of waste P.pastoris cells for Cu(II), Ni(II) and Cr(VI) in aqueous solution conditions. The results showed that the adsorption kinetics of three kinds of metals were well-fitted with lineared Elovich, pseudo-second-order kinetics models, non-linear kinetics and adsorption isotherms. The effective biosorption rates for Cu(II), Ni(II) and Cr(VI) removal were 71.3%, 59.7% and 16.25% respectively. The maximum Cu(II) adsorption capacity of waste P.pastoris was 40 mg/g at pH = 4 and 225 mg/L of solute concentration for 0.4 g biomass, better than that of the living yeasts. The pattern of Fourier transform infrared (FTIR) indicated that functional groups such as -NH, -OH, Si-O, P-O-C were involved in Cu(II) adsorption process. The analysis of SEM-EDS, XRD and TEM-EDS can be concluded that Cu(II) occupied Ca(II) binding sites by ion exchange mechanism to remove flocculation, and Cu(II) adsorbed onto the diatomite containing in the industrial waste P.pastoris. Thus the adsorption mechanism of the industrial waste P.pastoris was proposed taking Cu(II) as the example. And consecutive biosorption/desorption cycles were used for the evaluation of the regeneration efficiency, suggesting the good regeneration and reusability of waste P.pastoris.

Enhancing lactose recognition of a key enzyme in 2'-fucosyllactose synthesis: α-1,2-fucosyltransferase.

BACKGROUND: 2'-Fucosyllactose, a representative oligosaccharide in human milk, is an emerging and promising food and pharmaceutical ingredient due to its powerful health benefits, such as participating in immune regulation, regulation of intestinal flora, etc. To enable economically viable production of 2'-fucosyllactose, different biosynthesis strategies using precursors and pathway enzymes have been developed. The α-1,2-fucosyltransferases are an essential part involved in these strategies, but their strict substrate selectivity and unsatisfactory substrate tolerance are one of the key roadblocks limiting biosynthesis. RESULTS: To tackle this issue, a semi-rational manipulation combining computer-aided designing and screening with biochemical experiments were adopted. The mutant had a 100-fold increase in catalytic efficiency compared to the wild-type. The highest 2'-fucosyllactose yield was up to 0.65 mol mol-1 lactose with a productivity of 2.56 g mL-1  h-1 performed by enzymatic catalysis in vitro. Further analysis revealed that the interactions between the mutant and substrates were reduced. The crucial contributions of wild-type and mutant to substrate recognition ability were closely related to their distinct phylotypes in terms of amino acid preference. CONCLUSION: It is envisioned that the engineered α-1,2-fucosyltransferase could be harnessed to relieve constraints imposed on the bioproduction of 2'-fucosyllactose and lay a theoretical foundation for elucidating the substrate recognition mechanisms of fucosyltransferases. © 2022 Society of Chemical Industry.

Temperature mediated the balance between stochastic and deterministic processes and reoccurrence of microbial community during treating aniline wastewater.

Seasonal temperature changes significantly affect microbial community diversity, composition, and performance in wastewater treatment plants. However, the community assembly mechanisms under seasonal temperature variations remain unclear. Here, we carried out temperature cycling experiments (30 °C, 35 °C, 37 °C, 40 °C, 42 °C, 45 °C, 40 °C, and 30 °C) to investigate how temperature impacts microbial performance and co-occurrence network and how assembly processes determine the structure and function of microbial communities during treating aniline wastewater. During the 195-day operation, the system achieved an efficient and stable aniline removal of 99%. Interestingly, α-diversity and network complexity were negatively correlated with temperature but could be recovered when the temperature was returned to 30 °C. The results showed that functional redundancy was probably responsible for the excellent microbial performance during the whole process. Null model analyses presented that deterministic process dominated the community when the temperature was 30 °C, and stochasticity dominated the assembly process when the temperature was over 30 °C. Overall, the balance between stochastic and deterministic processes in the treatment of aniline wastewater mediated the reoccurrence of microbial community and co-occurrence network at different temperatures. This study provides new insights into microbial community reoccurrence under seasonal temperature changes and a theoretical basis for regulating microbial communities in wastewater treatment plants.

ARTP and NTG compound mutations improved Cry protein production and virulence of Bacillus thuringiensis X023.

A high production mutated strain Bacillus thuringiensis X023PN (BtX023PN) was screened from the wild strain Bacillus thuringiensis X023 (BtX023) after atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG) mutation. BtX023PN grows faster than the wild strain, and its lysis of mother cell was 6 h ahead BtX023, but the ability of sporulation was significantly reduced. Bioassay indicated that compared with the wild type strain, the virulence of BtX023PN against Plutella xylostella (P. xylostella) and Mythimna seperata (M. seperata) increased to 2.33-fold and 2.13-fold respectively. qRT-PCR and SDS-PAGE demonstrated that the production of Cry1Ac increased by 61%. Resequence indicated that the mutated sites enriched on the key carbohydrate metabolism and amino acid metabolism. This study provides a new strain resource for the development of Bt insecticides and a feasible technical strategy for the breeding of Bt. KEY POINTS: • Atmospheric and room temperature plasma used in breeding of Bacillus thuringiensis. • Less stationary phase time with more ICP production. • Semi-lethal concentration against Plutella xylostella reduced by about 57.

Enhancing heterologous expression of a key enzyme for the biosynthesis of 2'-fucosyllactose.

BACKGROUND: 2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide (HMO) in human milk and has important physiological functions. The market demand of 2'-FL is continuing to grow, but high production cost has limited its availability. To solve the dilemma, biosynthesis of 2'-FL has been proposed and is considered the most promising pathway for massive production. α-1,2-Fucosyltransferase is one of the key elements involved in its biosynthesis, but the limited intracellular accumulation and unstable properties of α-1,2-fucosyltransferases when expressed in host strains have become a major hurdle for the effective biosynthesis of 2'-FL. RESULTS: A combinatorial engineering strategy of synergic modification of ribosome binding site, fusion peptide and enzyme gene was leveraged to enhance the soluble expression of α-1,2-fucosyltransferases and promote enzyme activity. The preferable combination was to employ an optimized ribosome binding site region to drive 3 × FLAG as a fusion partner along with the α-1,2-fucosyltransferase for expression in Escherichia coli (DE3) PlySs, and protein yield and enzyme activity were remarkably improved by 11.51-fold and 13.72-fold, respectively. CONCLUSION: After finely tuning the synergy among different elements, the abundant protein yield and high enzyme activity confirmed that the drawbacks of heterologous expression in α-1,2-fucosyltransferase had been properly addressed. A suitable external environment further drives the efficient synthesis of α-1,2-fucosyltransferases. To our knowledge, this is the first report of a systematic and effective modification of α-1,2-fucosyltransferase expression, which could potentially serve as a guideline for industrial application. © 2022 Society of Chemical Industry.

Extracting copper and cobalt from non-ferrous residues by iron- and sulfur-oxidizing bacteria.

How to recycle metals from the waste resources becomes a hotspot all around the world. Non-ferrous residues, which was produced by non-ferrous melting industry, and various of Cu and Co compounds exist in the residues in the form of CuxOy, CuxSy, CoxSy. In order to efficiently extract valuable metals from the non-ferrous residues, this study investigated the bioleaching behavior of Cu and Co from non-ferrous residues, using iron-oxidizing bacteria (IOB, Leptospirillum ferriphilum CS13) and sulfur-oxidizing bacteria (SOB, Acidithiobacillus caldus S2) by controlling the microbial composition, initial pH, and initial ferrous ion concentration. The results showed that IOB had a better performance on extracting Cu and Co than that of SOB, especially for Cu. Furthermore, 77.7 and 79.8% of Cu and Co were extracted under the optimal ratio of the initial number of IOB and SOB (1:1) after bioleaching, which was more than that when bioleaching by any one of these two kinds of bacteria. However, the changes of initial pH and ferrous ion concentration could not significantly enhance bioleaching performance. The results indicated that bioleaching had a good performance on recovering of metals from non-ferrous residues and excellent application prospect for the cleaner resource recycling.

Organic acid leaching was an efficient approach for detoxification of metal-containing plant incineration ash.

Metal-containing plant incineration ash (MPIA), which was the by-product for metal extraction from soil by phytoextraction process, contains various kinds of heavy metal that have post potential risk to the environment. This study investigated the leaching efficiency and metal redistribution of MPIA using organic acid as leaching agents. The MPIA before and after leaching was characterized using the toxicity characteristic leaching procedure (TCPL) test, X-ray diffraction, scanning electronic microscopy, and Fourier transform infrared spectroscopy. The results showed that all tested organic acids resulted in the dissolution of metals, especially 1 mol L-1 citric acid leaching achieved for the dissolution efficiency of 84% Mn, 87.01% Cd, 66.97% Zn, and 55.83% Pb. During leaching progress, the synergetic of chelation and acid soluble action accelerated the metal release and redistribution, and the dissolution of Mn, Zn, Pb, and Cd fit best to the shrinking core model of chemical control. Meanwhile, the leaching residue reached the regulatory standard. Thus, organic acid leaching may be a feasible strategy for detoxification of MPIA.

Bioleaching of Copper-Containing Electroplating Sludge.

The recovery of precious metals from solid waste through bioleaching has become a research hotspot in recent years. Thus, in this study, different strategies, such as chemical sulfuric acid leaching and mixed consortium bioleaching, were adopted to extract copper from Copper-Containing Electroplating Sludge. The results showed that, compared to chemical leaching, bioleaching showed a much better performance. Indeed, copper bioleaching efficiency reached 94.3% on day 7 (21.1% higher than that of chemical leaching). The results also indicated that the process of bioleaching involved more mechanisms and reactions than that of chemical leaching. The SEM and EDX tests showed that the surface morphology of the sludge changed significantly after bioleaching, and that an insignificant amount of copper remained in the leached residues. Furthermore, the leached residues passed the characteristic leaching toxic test and thus can be considered as non-hazardous raw materials for the construction industry. Hence, adopting a mixed consortium leaching process to extract copper from Copper-Containing Electroplating Sludge will not only significantly reduce environmental pollution, but will also use metal resources more efficiently.

Adsorption process and mechanism of heavy metal ions by different components of cells, using yeast (Pichia pastoris) and Cu2+ as biosorption models.

Microbial biomass has been recognized as an essential biosorbent to remove heavy metal ions, but the biosorption process and mechanism of different components of microbial cells have not been elucidated. In present study, Pichia pastoris X33 and Cu2+ was used as a biosorption model to reveal the biosorption process and mechanism of different components of microbial cells. For the biosorption of whole cells, the maximum removal efficiency was 41.1%, and the adsorption capacity was 6.2 mg g-1. TEM-EDX analysis proved the existence of Cu2+ on the cell surface and cytoplasm. The maximum Cu2+ removal efficiency of the cell wall, cell membrane and cytoplasm were 21.2%, 20.7% and 18.5%, respectively. The optimum pH of Cu2+ biosorption of the P. pastoris cell, cell wall, cell membrane and cytoplasm was 6. Moreover, the maximum adsorption capacity of the cell, cell wall, cell membrane and cytoplasm was 16.13, 11.53, 10.97 and 8.87 mg g-1, respectively. The maximum removal efficiencies of P. pastoris biomass treated with proteinase K and P. pastoris biomass treated with ß-mannanase were 18.1% and 28.2%, respectively. The maximum removal efficiencies of mannan and glucan were 34% and 12%, respectively. The FTIR spectra showed that the amino group (N-H), hydroxyl (O-H), carbon oxygen bond (C-O), -CH, C-N and carbonyl group (C[double bond, length as m-dash]O) of a ketone or aldehyde may interact with Cu2+. Thus, our work provides guidance for further understanding the effect of different cell components on biosorption.

Efficient reduction of reactive black 5 and Cr(â ¥) by a newly isolated bacterium of Ochrobactrum anthropi.

It is important to obtain bacteria with the ability for reduction of dyes and Cr(Ⅵ) since dyes and Cr(Ⅵ) are often co-exist in textile wastewater. In this study, a new strain belonging to Ochrobactrum anthropi was isolated from textile wastewater, and could efficiently reduce Reactive Black 5 (RB 5) and Cr(Ⅵ). The results showed the degradation efficiency of RB 5 could achieve 100% and reduction efficiency of Cr(Ⅵ) was up to 80% within 3 days at initial RB 5 and Cr(Ⅵ) concentration of 400 mg/L and 20 mg/L. Mn2+ and Cu2+ could enhance the removal of RB 5 and Cr(Ⅵ), respectively. Glycerin, as electron donor, improved reduction efficiencies of RB 5 and Cr(Ⅵ). In addition, reduction mechanisms were further investigated. The results demonstrated that decreasing of RB 5 and Cr(Ⅵ) concentration were mainly through extracellular bioreduction rather than by adsorption. The FTIR and XPS analyses revealed that the O‒H, C‒C and C‒H groups on the cell surface might be involved in the reduction of RB 5 and Cr(Ⅵ). The information gives useful insights into understanding of how the bacterium reduce RB 5 and Cr(Ⅵ). The results indicated that the strain had excellent application prospect for treating industrial wastewater.

Isolation and characterization of lactic acid bacteria from human milk.

Human milk is the main source of nutrition for infants and the transmission of various microorganisms. The lactic acid bacteria (LAB) in breast milk allow for the establishment of the gut microflora of infants. In this study, we aimed to assess the probiotic potential of LAB strains isolated from breast milk of healthy Chinese women. Two strains, Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) LHL6 and LHL7, were selected and identified through morphology observation, Gram staining, and 16S rDNA phylogenetic analysis. Using Limosilactobacillus fermentum (formerly Lactobacillus fermentum) CECT5716 as the standard reference strain, the screened strains were characterized for aspects of growth, production of lactic acid and H2O2, antibiotic susceptibility, survival under simulated gastrointestinal conditions, and tolerance to cadmium (Cd). In de Man, Rogosa, and Sharpe (MRS) broth, LHL6 and LHL7 showed longer lag phases than CECT5716 but higher specific growth rates. For the production of lactic acid and H2O2, LHL7 performed better than LHL6 and CECT5716, indicating better antimicrobial ability. Strain LHL7 generated 9.99 mg/L H2O2, considerably higher than 1.25 mg/L for LHL6 and 2.33 mg/L for CECT5716. According to European Food Safety Authority minimum inhibitory concentrations, all of the investigated strains were resistant to chloramphenicol, streptomycin, and kanamycin. However, unlike LHL6 and CECT5716, LHL7 was susceptible to ampicillin and resistant to tetracycline. Resistance to azithromycin, cephalexin, and penicillin G were similar for all 3 strains, whereas CECT5716 was resistant to a higher concentration of roxithromycin. All 3 strains were able to survive in a simulated gastric-like solution, but a low percentage survived in the presence of 0.4% bile salt and 7% pancreatin. Encapsulation with protectants may enhance the survival rate. All 3 strains were tolerant to 500 mg/L Cd in MRS broth and to 1,000 mg/L Cd on MRS agar medium. In summary, 2 novel strains of LAB were obtained that have similar characteristics to the reference strain CECT5716. This work identified potential probiotic candidates for application in the food and pharmaceutical industries and facilitated identification of further probiotics.

Overexpression of an endogenous raw starch digesting mesophilic α-amylase gene in Bacillus amyloliquefaciens Z3 by in vitro methylation protocol.

BACKGROUND: Mesophilic α-amylases function effectively at low temperatures with high rates of catalysis and require less energy for starch hydrolysis. Bacillus amyloliquefaciens is an essential producer of mesophilic α-amylases. However, because of the existence of the restriction-modification system, introducing exogenous DNAs into wild-type B. amyloliquefaciens is especially tricky. RESULTS: α-Amylase producer B. amyloliquefaciens strain Z3 was screened and used as host for endogenous α-amylase gene expression. In vitro methylation was performed in recombinant plasmid pWB980-amyZ3. With the in vitro methylation, the transformation efficiency was increased to 0.96 × 102 colony-forming units µg-1 plasmid DNA. A positive transformant BAZ3-16 with the highest α-amylase secreting capacity was chosen for further experiments. The α-amylase activity of strain BAZ3-16 reached 288.70 ± 16.15 U mL-1 in the flask and 386.03 ± 16.25 U mL-1 in the 5-L stirred-tank fermenter, respectively. The Bacillus amyloliquefaciens Z3 expression system shows excellent genetic stability and high-level extracellular production of the target protein. Moreover, the synergistic interaction of AmyZ3 with amyloglucosidase was determined during the hydrolysis of raw starch. The hydrolysis degree reached 92.34 ± 3.41% for 100 g L-1 raw corn starch and 81.30 ± 2.92% for 100 g L-1 raw cassava starch after 24 h, respectively. CONCLUSION: Methylation of the plasmid DNA removes a substantial barrier for transformation of B. amyloliquefaciens strain Z3. Furthermore, the exceptional ability to hydrolyze starch makes α-amylase AmyZ3 and strain BAZ3-16 valuable in the starch industry. © 2020 Society of Chemical Industry.