Carotenoid productivity in human intestinal bacteria Eubacterium limosum and Leuconostoc mesenteroides with functional analysis of their carotenoid biosynthesis genes

The human intestinal microbiota that comprise over 1,000 species thrive in dark and anaerobic environments.They are recognized for the production of diverse low-molecular-weight metabolites crucial to human health and diseases.Carotenoids,low-molecular-weight pigments known for their antioxidative activity,are delivered to humans through oral intake.However,it remains unclear whether human intestinal bacteria biosynthesize carotenoids as part of the in-situ microbiota.In this study,we investigated carotenoid synthesis genes in vari-ous human gut and probiotic bacteria.As a result,novel candidates,the crtM and crtN genes,were identified in the carbon monoxide-utilizing gut anaerobe Eubacterium limosum and the lactic acid bacterium Leuconostoc mesenteroides subsp.mesenteroides.These gene candidates were isolated,introduced into Escherichia coli,which synthesized a carotenoid substrate,and cultured aerobically.Structural analysis of the resulting carotenoids re-vealed that the crtM and crtN gene candidates of E.limosum and L.mesenteroides mediate the production of 4,4′-diaponeurosporene through 15-cis-4,4′-diapophytoene.Evaluation of the crtE-homologous genes in these bacteria indicated their non-functionality for C40-carotenoid production.E.limosum and L.mesenteroides,along with the known carotenogenic lactic acid bacterium Lactiplantibacillus plantarum,were observed to produce no carotenoids under strictly anaerobic conditions.The two lactic acid bacteria synthesized detectable levels of 4,4′-diaponeurosporene under semi-aerobic conditions.The findings highlight that the obligate anaerobe E.limo-sum retains aerobically functional C30-carotenoid biosynthesis genes,potentially with no immediate self-utility,suggesting an evolutionary direction in carotenoid biosynthesis.(229 words)

Spatiotemporal variations of sand hydraulic conductivity by microbial application methods

The spatiotemporal distributions of microbes in soil by different methods could affect the efficacy of the microbes to reduce the soil hydraulic conductivity.In this study,the specimens of bio-mediated sands were prepared using three different methods,i.e.injecting,mixing,and pouring a given microbial so-lution onto compacted sand specimens.The hydraulic conductivity was measured by constant-head tests,while any soil microstructural changes due to addition of the microbes were observed by scan-ning electron microscope(SEM)and mercury intrusion porosimetry(MIP)tests.The amount of dextran concentration produced by microbes in each type of specimen was quantified by a refractometer.Results show that dextran production increased exponentially after 5-7 d of microbial settling with the supply of culture medium.The injection and mixing methods resulted in a similar amount and uniform dis-tribution of dextran in the specimens.The pouring method,however,produced a nonuniform distri-bution,with a higher concentration near the specimen surface.As the supply of culture medium discontinued,the dextran content near the surface produced by the pouring method decreased dramatically due to high competition for nutrients with foreign colonies.Average dextran concentration was negatively and correlated with hydraulic conductivity of bio-mediated soils exponentially,due to the clogging of large soil pores by dextran.The hydraulic conductivity of the injection and mixing cases did not change significantly when the supply of culture medium was absent.

酿酒酵母产D-乳酸重组菌的构建与发酵

采用基因工程方法对酿酒酵母进行代谢改造,使酵母产生乳酸代谢途径。将来源于L.mesenteroides和E.coli的D-乳酸脱氢酶基因,分别插入带有G418抗性的酵母穿梭质粒p YX212-kan MX上,电转化酵母,得到2株生产D-乳酸的酿酒酵母重组菌S.cerevisiae WE1510和S.cerevisiae WB1186。进一步摇瓶发酵试验表明:重组菌S.cerevisiae WB1186在YEPD培养基、20 g/L糖、p H 5的条件下生长条件最好,并具有更好的产乳酸能力。经3 L发酵罐条件下验证,S.cerevisiae WB1186分批发酵96 h,最终乳酸积累量达到18.0 g/L;发酵条件为培养基YEPD,接种量10%,溶解氧(DO)30%,转速150 r/min,初始葡萄糖质量浓度10 g/L,控制pH 5.0,通气量3 L/min,OD600最大值转为厌氧发酵。

Study on Lysine and Methionine Content Promotion of Soybean Meal by Probiotic Fermentation Process

Soybean meal （SBM） is commonly used for livestock feeds, but its application in diets for livestock is limited due to some antinutritional factors. The contents of methionine and lysine of soybean meal were promoted by Bacillus natto and Leuconostoc mesenteroides fermentation, benefial for the livestock feeds. It was crude protein （CP） 56.8%, methionine 43.56 mg · g^-1, and lysine 74.87 mg · g^-1, cows fed a diet with FSBM milk yield raised 14.2%, the change in the milk protein, the lactose and the dry matter content had also obvious increase. This convenient technique offers helpful exploration for industrialization of soybean meal fermentation.

Permeability and setting time of bio-mediated soil under various medium concentrations

The bio-clogging using bacteria can be an eco-friendly and sustainable alternative to conventional grouting methods for seepage control.However,it remains unclear to date how the dilute concentration of bacterium and medium during field installation can affect the setting time of bacterium and its correlation with permeability reduction.In this study,the setting time of bacterium and its effectiveness in permeability reduction were addressed through experimental and theoretical investigations.A series of sand column was cultivated using different concentrations of Leuconostoc mesenteroides and culture medium.The distribution and composition of the bacterial product(i.e.dextran)were observed by refractometer,scanning electron microscope(SEM),and energy dispersive X-ray spectroscopy(EDS).Soil permeability was recorded using a constant head test.The results revealed that bacterium was effective to produce dextran at the setting time of about 5 d after installation.This dextran can reduce the permeability of bio-mediated soil by two orders of magnitude,even without culture medium supply.In general,the dextran production decreased proportionally with increase of bacterium and medium concentration.However,at 50%bacterium and medium concentration by weight,it still has a significant influence on permeability reduction with similar setting time,compared to 100%concentration.

Application of Protein Feed Processed by Microbial Fermentation to Dairy Cow

Methionine （Met） and lysine （Lys） have been reported as the first two limiting amino acids （AA） for maximum milk yield and milk protein production. Supplying these AA may improve microbial protein synthesis and therefore improve milk production without adding excess N to the environment. This observation utilized fermented soybean meal （SBM）, cottonseed meal （CSM）, rapeseed meal （RSM） and corn by Bacillus subtilis 168 and Leuconostoc mesenteroides as core feedstuffs to produce special biological protein feed for dairy cow. The results showed that the milk production, milk protein percentage, milk fat percentage and milk DM percentage of test groups in trial period were significantly more than those of the control group （P〈0.01）, the results showed that adding fermenting protein feed in dairy cow diets could significantly improve milk yield, milk protein and milk fat content. The economic benefits of actual application were analyzed, the group of 0.5% was the best compared with the other groups.

辣椒酱发酵菌肠膜明串珠菌C27高密度培养条件优化

为了实现辣椒酱发酵肠膜明串珠菌(Leuconostoc mesenteroides)C27的高密度培养,以MRS肉汤培养基为基础,L.mesenteroides C27的菌体密度为评价指标,采用单因素试验和响应面法对培养基中的碳源、氮源、生长因子进行优化,同时采用响应面法对培养条件进行优化。结果表明,最佳培养基配方为蔗糖21 g/L,酵母浸粉22 g/L,土豆汁14 g/L;最佳培养条件为发酵温度38.4℃、初始pH值6.2,接种量2.4%。在此优化条件下培养24 h,L.mesenteroides C27的菌体密度(OD600 nm值)达1.034,活菌数为1.30×109 CFU/mL。