Resazurin as an indicator of reducing capacity for analyzing the physiologic status of deep-sea bacterium Photobacterium phosphoreum ANT-2200

Resazurin(RZ)is a weakly fl uorescent blue dye and can be reduced irreversibly to highly fl uorescent pink resorufi n(RF)that is reduced reversibly to colorless dihydroresorufi n(hRF)by photodeoxygenation,chemical reaction and reductive organic compounds produced through cell metabolism.Because of the reliable and sensitive fl uorescence-color change and noninvasive features,RZ has been used widely as a redox indicator in cell viability/proliferation assays for bacteria,yeast,and mammalian cells.However,RZ is used rarely for physiological characterization of marine microorganisms.Here,we developed a custom-made irradiation and absorption-analysis device to assess the reducing capacity and physiologic status of marine bacterial cultures.We measured the absorption spectra of RZ,RF,and hRF in the presence of the reducing compound Na 2 S and under visible-light irradiation.After establishing appropriate parameters,we monitored the color changes of RZ and its reduced derivatives to evaluate the coherence between reducing capacity,bioluminescence and growth of the deep-sea bacterium Photobacterium phosphoreum strain ANT-2200 under various conditions.Emission of bioluminescence is an oxidation process dependent upon cellular reducing capacity.Growth and bioluminescence of ANT-2200 cell cultures were impeded progressively with increasing concentrations of RZ,which suggested competition for reducing molecules between RZ at high concentration with reductive metabolism.Therefore,caution should be applied upon direct addition of RZ to growth media to monitor redox reactions in cell cultures.Analyses of the instantaneous reduction velocity of RZ in ANT-2200 cell cultures showed a detrimental eff ect of high hydrostatic pressure and high coherence between the reducing capacity and bioluminescence of cultures.These data clearly demonstrate the potential of using RZ to characterize the microbial metabolism and physiology of marine bacteria.

Environment and synthetic biology

Industrial expansion and population growth stimulates the utilization of natural resources and energy.It inevitably leads to environmental pollution with various pollutants like heavy metals,pesticide and xenobiotics.Synthetic biology is an interdisciplinary field involving designation,construction,and manipulation of biological parts or devices in chassis and organisms for diverse purposes.It’s a promising alternative for synthetic biotechnology to overcome the global environmental issues by engineering microorganisms.

Microbial production of cis,cis-muconic acid from aromatic compounds in engineered Pseudomonas

Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons.Pseudomonas spp.have broad metabolic potential for degrading aromatic compounds.The objective of this study was to develop a“biological funneling”strategy based on genetic modification to convert complex aromatic compounds into cis,cis-muconate(ccMA)using Pseudomonas putida B6-2 and P.brassicacearum MPDS as biocatalysts.The engineered strains B6-2(B6-2ΔcatBΔsalC)and MPDS(MPDSΔsalC(pUCP18k-catA))thrived with biphenyl or naphthalene as the sole carbon source and produced ccMA,attaining molar conversions of 95.3%(ccMA/biphenyl)and 100%(ccMA/naphthalene).Under mixed substrates,B6-2ΔcatBΔsalC grew on biphenyl as a carbon source and transformed ccMA from non-growth substrates benzoate or salicylate to obtain higher product concentration.Inserting exogenous clusters like bedDC1C2AB and xylCMAB allowed B6-2 recombinant strains to convert benzene and toluene to ccMA.In mixed substrates,constructed consortia of engineered strains B6-2 and MPDS specialized in catabolism of biphenyl and naphthalene;the highest molar conversion rate of ccMA from mixed substrates was 85.2%when B6-2ΔcatBΔsalC was added after 24 h of MPDSΔsalC(pUCP18k-catA)incubation with biphenyl and naphthalene.This study provides worthwhile insights into efficient production of ccMA from aromatic hydrocarbons by reusing complex pollutants.

复合菌系降解纤维素过程中微生物群落结构的变化

为明确高效纤维素降解复合菌系降解过程中微生物群落结构的变化规律及关键的降解功能菌,利用该复合菌系对滤纸和稻秆进行生物处理,通过底物降解、微生物生长量、发酵液pH的变化情况,选择不同降解时期复合菌系提取的总DNA进行细菌16S rRNA基因扩增子高通量测序。通过分解特性试验确定在接种后培养第12、72、168 h分别作为降解初期、高峰期、末期。该复合菌系分别主要由1个门、2个纲、2个目、7个科、11个属组成。随着降解的进行,短芽胞杆菌属Brevibacillus、喜热菌属Caloramator的相对丰度逐渐降低;梭菌属Clostridium、芽胞杆菌属Bacillus、地芽胞杆菌属Geobacillus、柯恩氏菌属Cohnella的相对丰度逐渐升高;解脲芽胞杆菌属Ureibacillus、泰氏菌属Tissierella、刺尾鱼菌属Epulopiscium在降解高峰期时相对丰度最高;各时期类芽胞杆菌属Paenibacillus、瘤胃球菌属Ruminococcus的相对丰度无明显变化。上述11个主要菌属均属于厚壁菌门,具有嗜热、耐热、适应广泛pH、降解纤维素或半纤维素的特性。好氧型细菌是降解初期的主要优势功能菌,到中后期厌氧型细菌逐渐增多,并逐步取代好氧型细菌成为降解纤维素的主要细菌。

Biodegradation of aromatic pollutants meets synthetic biology

Ubiquitously distributed microorganisms are natural decomposers of environmental pollutants.However,because of continuous generation of novel recalcitrant pollutants due to human activities,it is difficult,if not impossible,for microbes to acquire novel degradation mechanisms through natural evolution.Synthetic biology provides tools to engineer,transform or even re-synthesize an organism purposefully,accelerating transition from unable to able,inefficient to efficient degradation of given pollutants,and therefore,providing new solutions for environmental bioremediation.In this review,we described the pipeline to build chassis cells for the treatment of aromatic pollutants,and presented a proposal to design microbes with emphasis on the strategies applied to modify the target organism at different level.Finally,we discussed challenges and opportunities for future research in this field.

Pollution and biodegradation of hexabromocyclododecanes:A review

Hexabromocyclododecanes(HBCDs)are the most common brominated flame-retardants after polybrominated diphenyl ethers.HBCDs can induce cancer by causing inappropriate antidiuretic hormone syndrome.Environmental contamination with HBCDs has been detected globally,with concentrations ranging from ng to|ig.Methods to degrade HBCDs include physicochemical methods,bioremediation,and phytoremediation.The photodegradation of HBCDs using simulated sunlight or ultraviolet lamps,or chemical catalysts are inefficient and expensive,as is physicochemical degradation.Consequently,bioremediation is considered as the most cost-effective and clean approach.To date,five bacterial strains capable of degrading HBCDs have been isolated and identified:Pseudomonas sp.HB01,Bacillus sp.HBCD-sjtu,Achromobacter sp.HBCD-1,Achromobacter sp.HBCD-2,and Pseudomonas aeruginosa HS9.The molecular mechanisms of biodegradation of HBCDs are discussed in this review.New microbial resources should be explored to increase the resource library in order to identify more HBCD-degrading microbes and functional genes.Synthetic biology methods may be exploited to accelerate the biodegradation capability of existing bacteria,including modification of the degrading strains or functional enzymes,and artificial construction of the degradation microflora.The most potentially useful method is combining microdegradation with physicochemical methods and phytoremediation.For example,exogenous microorganisms might be used to stimulate the adsorption capability of plants for HBCDs,or to utilize an interaction between exogenous microorganisms and rhizosphere microorganisms to form a new rhizosphere microbial community to enhance the biodegradation and absorption of HBCDs.

Guided by the principles of microbiome engineering:Accomplishments and perspectives for environmental use

Although the accomplishments of microbiome engineering highlight its significance for the targeted manipulation of microbial communities,knowledge and technical gaps still limit the applications of microbiome engineering in biotechnology,especially for environmental use.Addressing the environmental challenges of refractory pollutants and fluctuating environmental conditions requires an adequate understanding of the theoretical achievements and practical applications of microbiome engineering.Here,we review recent cutting-edge studies on microbiome engineering strategies and their classical applications in bioremediation.Moreover,a framework is summarized for combining both top-down and bottom-up approaches in microbiome engineering toward improved applications.A strategy to engineer microbiomes for environmental use,which avoids the build-up of toxic intermediates that pose a risk to human health,is suggested.We anticipate that the highlighted framework and strategy will be beneficial for engineering microbiomes to address difficult environmental challenges such as degrading multiple refractory pollutants and sustain the performance of engineered microbiomes in situ with indigenous microorganisms under fluctuating conditions.

Unveiling degradation mechanism of PAHs by a Sphingobium strain from a microbial consortium

Polycyclic aromatic hydrocarbons(PAHs)are a class of persistent pollutants with adverse biological effects and pose a serious threat to ecological environments and human health.The previously isolated phenanthrene‐degrading bacterial consortium(PDMC)consists of the genera Sphingobium and Pseudomonas and can degrade a wide range of PAHs.To identify the degradation mechanism of PAHs in the consortium PDMC,metagenomic binning was conducted and a Sphingomonadales assembly genome with 100%completeness was obtained.Additionally,Sphingobium sp.SHPJ‐2,an efficient degrader of PAHs,was successfully isolated from the consortium PDMC.Strain SHPJ‐2 has powerful degrading abilities and various degradation pathways of high‐molecular‐weight PAHs,including fluoranthene,pyrene,benzo[a]anthracene,and chrysene.Two ring‐hydroxylating dioxygenases,five cytochrome P450s,and a pair of electron transfer chains associated with PAH degradation in strain SHPJ‐2,which share 83.0%–99.0%similarity with their corresponding homologous proteins,were identified by a combination of Sphingomonadales assembly genome annotation,reverse‐transcription quantitative polymerase chain reaction and heterologous expression.Furthermore,when coexpressed in Escherichia coli BL21(DE3)with the appropriate electron transfer chain,PhnA1B1 could effectively degrade chrysene and benzo[a]anthracene,while PhnA2B2 degrade fluoranthene.Altogether,these results provide a comprehensive assessment of strain SHPJ‐2 and contribute to a better understanding of the molecular mechanism responsible for the PAH degradation.