Exploiting microbial ecology

A/ CanadaProf A. Mulchandani (University of California), Prof J. Zhou (University of Oklahoma),Global market values for enzymes in biocatalysis10307802507030V a l u e s i n m i l l i o n ($)Global values for enzymes in bioconversion Pharmaceuticals Agrochemicals2500010000V a l u e s i n m i l l i o n ($)Global Markets for Microbial products Singh et al 2010 Trend BiotechnolTotal industrial markets for microbial enzymes: several trillions/ yearGlobal markets of remediationTotal global market is estimated at US $ 1 trillionSource: Singh et al. 2009 124.85550313124*Also includes other environmental industriesWhy remediation/ bioremediation?•Regulatory requirements•Sustainable development•Re-use of land•Commercial benefitsTypes of remediation technologies1.Biological processes: microbial remediation,phytoremediation, bioventing2.Chemical processes: Oxidising agents, chemicalextractions3.Physical processes: Soil washing, vapour extractions4.Thermal processes: Incineration, supercritical oxidationTypes of bioremediation technologies 1. Microbial bioremediation: Bacteria, fungi, archaea and theirproductsA. BioaugmentationB. BiostimulationC. Intrinsic bioremediation2. Phytoremediation:Plant assistedbioremediationBioremediation Some success storiesOP compounds and problemsLeaving groupOPH enzymeSingh , BK (2009). Nature Rev. Microbiol.1.~ 38% of total pesticide usages2.World-wide usageed in large quantities4.Extreme mammalian toxicity &structurally similar to nerve agents OP (Organophosphorus compounds)OP compounds and medical problems 1.3 Million poisoning incidence-Medicalproblem.2.200,000 death per year due to OP–poisoning3.Lot of un-used OP Chemical warfareagents as potential source for terroristattacksNo effective treatment availablefor overwhelming poisoningSingh and Walker, 2006. FEMs Microbiol. Rev.Eddleston et al., 2008, LancetOP and sources of contamination and toxicitySources of acute toxicity1.Manufacturing wasterge scale use e.g. Cattle dips3.Accidental spillage4.Terrorist attacksSmall release and chronic effects 1.Spraying2.Cleaning of containers and equipment3.Bioaccumulation through food chainCurrent bioremediation technologies are inefficient, expensive and time consumingBiofilter for Coumaphos remediation in USA110100100010000025101820C o u m a p h s d e g r a d a t i o nDaysBiofilter Control•Comasphos waste generated from cattle dips (conc: 2000 mg/ litre)•11,000 litre biofilter has been successfully used for number of years for decontaminationMulbry et al; 1998, 20005101520250246810P a r a t h i o n (m g /k g )Parathion-control Parathion05101520250246810D i a z i n o n (m g /k g )Diazinon-control Diazinon05101520250246810C o u m a p h o s (m g /k g )Time (Days)Coumaphos-control Coumaphos5101520250246810I s a z o f o s (m g /k g )Time (Days)Isazofos-control IsazofosBioremediation in SoilsSingh et al., 2003; 2006But significant numbers of bioremediation approaches failWHY?of four different OP compounds by Enterobacter sp in soilsAbiotic properties Vs Microbial activitySoil pH V microbial activity•Compounds-chlorpyrifos & fenamiphos• 2 acidic, 2 alkaline and 1 neutral pH soilsy = 0.1993x -0.8401R² = 0.950.10.20.30.40.50.60.70.80.9456789o p d g e n e (R A )Soil pHSoil pH vs opd geney = 137.14e -5.216xR² = 0.9602040608010000.20.40.60.81C h l o r p y r o s (% r e s i d u e )opd gene abundanceChlorpyrifos Vs opd geneSoil abiotic vs gene Vs functionSingh (2009) Nat Rev MicrobiolDegradation pathway of OP compoundsLeaving groupOPH enzymeSingh and Walker 2006. FEMS-MRChlorpyrifosFenamiphosChemical structure and degradationBond cleavageBond cleavageKarpouzas and Singh 2006, AMPMicrobial diversityand xenobiotic degradationy = 804.04e -5.846xR² = 0.910204060801000.30.50.70.9C h e m i c a l A (% r e s i d u e )Relative diversity (Geochip)Diversity Vs function (biodegradation)Adapted from Singh et al 2003; 2009; 2014y = -138.5ln(x) -1.642R² = 0.49260204060801000.50.60.70.80.91C h e m i c a l A (% r e s i d u e )Relative diversity (16s DNA)Structural diversity vs function•Pyrosequencing (~20,000 seq)Functional diversity vs function•Geochip (>50,000 genes)These understandings have provided outstanding Successes-------Engineered Biopile Technology•Bioremediation of contaminated soils:•Bioaugmentation and Biostimunationn•Better control of oxygen, moisture, nutrient levels,pH and mixingA. Singh et al.,Examples of bioreactors operating atfull scaleExample of an integrated approachOily sludge management in oily pitsA three-phase centrifuge for oil recoveryOily waste pit Engineered Biopile for contaminated sandBioreactors for inoculum prep and oil sludgeOil extracted was worth more than the cost of remediationA Singh Lystek LtdConstraints associated withmicrobial bioremediation1.Sustaining a stable population of microorganisms2.Sustaining the activity of the microorganisms3.Other factors can influence activityAnother solution: use to Freedegrading enzymesAdvantage of using enzymes :1.Rapid decontamination2.No requirements of nutrients3.Theoretically could be re-suesDisadvantage:ck of enzyme diversity2.Problem with large scale production3.Low stability in fermenter/ biofilterck of mechanism to protect fromenvironmental protease204060801001200261018244872O P d e g r a d a t i o n HoursBacteria Enzymes ControlLandguard Tm is a good examples (A CSIRO + Orica product)Use of existing enzymes for bioremediation& industrial usages1. 1 g soil = ~104bacterial species = `1 million putative enzymes2.Soil contains 5 X 1030bacterial cells3.Environmental microbes areenormous source of new enzymesSingh, BK (2009). Nature Rev.Microbiol .1.Several microbes can degradeOPs but only 3 -5 types of enzymes 2. All enzymes from cultured bacteria(>99% microbes are unculturable)Solution from emerging and genomicsapproaches1.Metagenomics: For enzyme diversity2.Genome sequencing3.Metabolic Engineering: For enzyme and bioremediationefficiency4.Nanotechnology for stability and protectionOld approachOmic technologiesSlide: Hangwei HuOld technologies VS new technologiesBut microbes are enormously diverse and a conservative estimate suggests 2 millionclones/ sample need to be screenedSingh (2010). Trend. Biotechnol.MetagenomicsNow Before13 years $ 3 billion Human genome3.3x109 (10x coverage)8 days$ 4,000 Not possible Deep sequencingof somatic mutations(5000x coverage)60 days E. coli4.7x106 (5x coverage)14 months D. melanogaster1.4x108 (5x coverage) Improvements coming with the new sequencing technologies10 hours< 2 daysYES2007/20081-1.5Gb200810Gb200950Gb201095Gb1800Gb30x Coverage 18 Human Genomes=1 run HiSeq HxHiSeq HxGA/GA IIxIllumina output increased over thelast seven yearsConstraints:1. Bioinformatics support2. Loss of novel functions2011600Gb2014Soil from contaminated sitesStable isotope probingMetagenomic libraryScreen clones for OP degradation function12C DNA 13C DNASingh et al. (2010). Trend. Biotechnol.Nanotechnological formulationTherapeutic and prophylacticefficacyBioremedial efficacyFrom metagenome to applicationsDNA/RNA extraction12C DNA13C DNASeparation of 13C –12C nucleic acids inCsCl density centrifugationFractionationStable isotope probing (SIP)13C12CPulse label with 13CpesticideHarvest Singh (2010), Trend. Biotechnolo.Selective enrichment of DNA forpesticide degrading bacteria1.Isolation of five more efficientenzymes2.Success rates increase byseveral hundreds foldsSingh (2010). Trend. Biotechnol.SIP-Metagenomics13 nm40 nmEnzymeNano-porous mediaAbsorbed enzymes in nano-porous mediaCross-linked enzymes aggregated via GA treatment+NanotechnologyXProtease and mechanical sharing resistant enzymes for human treatment and bioremediation0204060801000123456P e s t i c i d e (%)Time (Hours)1st round 4th round0204060801000123456P e s t i c i d e (%)Time (Hours)1st round 4th round 25th roundFree-enzymesEnzymes in nano-porous structureNanotechnology and effects onre-usability of enzymesSignificant outcomesThis approach has already resulted a few novel enzymes for degradation which can be potentially used for1.Cheap and re-usable bioremediation technology for OP compounds2. A potential medical treatment for OP poisoning3.An effective prophylactic measures against OP poisoning4.Development of a rapid biosensor for OPcompoundsEarth is a microbial planet&Solutions to most tasks can be found in microbial communityThank youPhytoremediationFrom Ma et al (2010). BIOTECHNOLOGY ADVANCES, 28,367-374Problems with mixed organic and metalwastes•Forty percent of hazardous waste sites in the United States are co-contaminated with organic and metal pollutants•Metals affect organic biodegradation through impacting both the physiology and ecology of organic-degradingmicroorganismsEnvironmental Health Perspectives • VOLUME 111 | NUMBER 8 | June 2003BIOREMEDIATION IN THERHIZOSPHERECl -CO 2cell masscontaminantcontaminant-removing, root-colonizing bacteriaPCB, TCE, MetalsPHYTOREMEDIATION •Current problems associated with Phytoremediation•Plants Lack of growth due metal and organiccontaminationvolatilisation of organic compoundsstore them in leaves/shoots•This makes it less efficient and prevent using tree/ plants for any other purposesWe developed a novel technology called‘Designer Plants’•This technology brings multiple commercial and environmental benefits•A custom made solution for mix pollutants based on type and nature of contaminantsFungi RootsBacteriametalsOC 1OC 2OC 3Designer PlantsComplex level of contaminatione.g. mining siteAbhilash and Singh 2012 TIBIndustry Transport HeatingP y r o l y s i sB i o f u e lContaminated siteMixed: metals + organicAfforestation with bioenergy tree WoodBiomass energyAccelerated bioremediation & C sequestrationEnergy sourceMetal extractionEnhanced ecosystem Health & C farmingBenefits of ‘Designer Plants’Rapid pollutant removalBioremediation Designer plantsRayu & Singh 2012 Biodegrad。