Biochar as a Carrier for Microbial Inoculants

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Biochar as a Carrier for Microbial InoculantsBiochar as a Carrier for Microbial InoculantsDavid Crowley, Milt David Crowley, Milt McGiffenMcGiffen, Lauren Hale, Lauren HaleUniversity of California, RiversideUniversity of California, RiversideSoil Inoculation Technology0 AD Romans use soil from legumes to inoculate new fields.1896 Basic methods using peat as a carrier developed by Nobbe and Hiltner.1930s Large scale field inoculation with Azotobacter in Russia begun and then abandoned in 1950s. Likewise, use of Bacillus megaterium for phosphate solubilization begun on large scale in Eastern Europe, but failed.1950s Large scale use of Rhizobium inoculants begins, Widespread problems with contamination, inconsistent performance. 1970s Government agencies established to regulate inoculum quality established in Australia, USA, Canada1980s Mycorrhizal inoculants developed for ecosystem restoration.1990s Soil inoculation technologies extended for plant growth promoting microorganisms and bioremediation. Field trials mostly fail.2000New inoculation technologies. DNA based monitoring of inoculants.2010Commercialization and widespread use of microbial inoculants for agricultural biotechnology and environmental cleanup begins as serious global enterprise.Inoculated with PGPRControl - No PGPREnhanced Legume Growth following Inoculation with Plant Growth PromotingRhizobacteria (PGPR).Thousands of studies have shown the utility of soil inoculants for plant growthpromotion and bioremediation in the laboratory and the greenhouse. Field studies are less consistent due to poor understanding of soil ecology and monitoring of inoculant survival and activity.Trichoderma Inoculants for Plant Growth PromotionTrichoderma Inoculants for Plant Growth PromotionPlant Growth Promoting Bacteria: AzotobacterPlant Growth Promoting Bacteria: AzotobacterAzotobacter on Peanut (India)PGPR Soil Inoculant Factory China Agricultural UniversityBacillus crop enhancersImproved root growthwater use efficiencyfertilizer use efficiencyImproved soil structure Control of root diseases Bacillus cereusFunctions of Plant Growth Promoting Bacteria in the Rhizosphere Yang et al. 2009 Trends in Plant ScienceCharacterization of individual bacterial isolates for PGPR activitiesNadeem, Shaharoona, Crowley, Unpublished data 2011Copyright 2007 American Society of Plant BiologistsRuzicka, K., et al. Plant Cell 2007;19:2197-2212Ethylene Inhibition of Root Growth and Role of PGPR with ACC Deaminase ActivityACCPGPR BacteriaACC deaminase ketoglutarate+NH4Drought Tolerance and Long Term Selection of PGPR: The 11,700 Year-Old, King Clone of Larrea tridentaLucerne Valley, San Bernadino County, CaliforniaSampling of the Rhizosphere from the Worlds Oldest Living PlantThe King CloneLarrea tridenta(Creosote bush) Metagenomic Analysis of the Rhizosphere from the 11,700 Year Old King CloneCollection of Rhizosphere BacteriaJanuary 2010 Crowley & JorqueraProperties of Selected PGPR Isolates from the King Clone( ) = percent increase in comparison to sterilized control soilInoculant TechnologySeed inoculantsSoil Inoculum - Carrierspeat, coal, soil, calcined clay, vermiculite, perliterock phosphatepolymers, alginate beads,plant waste products (corn cobs)slurries, liquids, powdersBiochar?Desired characteristicsSterile carrier media, defined culturesHigh cell densityLong shelf life (months to years)Low costInoculum Production MethodsRodriguez-Navarro, Agron Sustain. Dev. 2010Inoculants and Nitrogen Fixation of Legumes in Vietnam, Herridge 2002Importance of Inoculum Density (Cell Numbers/Gram) for Rhizobium nodule formation and Relationship to Grain YieldsPotential Applications of Microbial Inoculants for Agricultural BiotechnologyPotential Applications of Microbial Inoculants for Agricultural BiotechnologyBiofertilizersMycorrhizaePhosphate solubilizing bacteriaPhosphate solubilizing fungiN fixing bacteriaSeleniumBiocontrolPlant root diseasesInsectsWeedsPlant Growth EnhancersRoot growth promotionHormone productionRemoval of cyanideEthylene suppressionBioremediationOilPesticidesMunitionsAzo dyesSolventsMetalsStudies in Japan in the 1990s show increases in plant nutrient uptake, mycorrhizae formation, and yields following inoculation with rhizobium and the addition of charcoal. of commercial products for Successful Use of Soil Inoculants:InoculumLow cell numbersContamination with undesired microorganismsHigh cost for culture mediaPoor survival in storageHigh cost of transporting inoculum to the fieldPractical methods to incorporate inocula into the soilPhysiologyStarvationPredationRapid cell deathLow activityEcologyCompetitive exclusion from the rhizospherePoor adaptation of inoculum to local soil conditions pHNo methods to easily monitor survival and activityThe BioJect system offers a novel approach to applying microbes - it grows them on site! The BioReactor unit is a fermentation vessel in which beneficial microbes multiply to high concentrations and are injected on a daily basis into the irrigation system for distribution over the entire course. In this way the BioJect is able to consistently treat large areas while conserving valuable labor resources. Adaptation of Batch Culture Bioreactor for Inoculation of Biochar: Custom Production of Microbial CulturesMethods for On-Site Automated Inoculum ProductionChanging Paradigms: Purpose of InoculationChanging Paradigms: Purpose of InoculationSingle and Mixed Inoculants:Introduce a missing function (eg N fixation), or to temporarily increase the population of plant growth promoting bacteria to an effective population size, generally 104 to 106 cells gramNew Paradigm :Shape the microbial community structure to promote optimal soil biological functions associated with plant health. Includes use of management practices, soil amendments (char and composts) and use of inoculants tailored to individual crops and soils. of Artificial Neural Network Models for Monitoring MicrobialCommunity Responses to Soil Management Practices (Biochar, Microbial Inoculants, Soil Type)Microbial community% ClaySoil pHBiocharDisease statusYieldsSampling: 34 fallow soilsconventional vs organic Microbiological Analyses: 58,000 16S rRNA gene sequencesIllumina high throughput sequencingsequences sorted by taxon (family)Survival of E. coliSoil Analyses:N, P, K, Ca,Management: organic vs conventionalpH, salinity, texture, bulk densityorganic carbon, total and water solubleArtificial Neural Network Analysis of Factors Affecting Microbial Community Structures in Arid Zone Agricultural Soils of SW USA (Ma, Ibekwe, Yang, Crowley, unpublished data)020004000600080001000012000140001600012100420063008400105001260014700168001890021000231002520027300294003150033600357003780039900420004410046200483005040052500546005670058800# of Sequences Sampled# of OTU's ObservedAZIMSASensitivity analysis of output variable to selected input variablesPrediction of dependent variable values in relation to all independent variablesEffects of Soil Variables on Microbial Community Structure on Arid Zone Agricultural Soils in Southern California3210.50.2140120100806040Predicted changes in Proteobacteria classes with water soluble organic carbon and salinitySummaryThe use of biochar as a soil amendment offers new opportunities to develop improved biofertilizer formulations that provide value-added enhancement of commercial biochar products.Research is in progress to determine optimal pyrolysis substrates and conditions to optimize biochar properties as an inoculum carrier. Much can be learned from previous research on rhizobium. Quality control and assurance requires testing of the efficacy of biochar products for modifying soil microbial community structures and PGPR populations. Decision support tools based on ANN models are now being developed to predict the effects of management practices, biochar amendments and soil inoculants on plant yields in different soils and climates. Development Process for Use of Biochar as an Inoculum Carrier Determine relationship between pyrolysis temperature, char properties, and suitability as a habitat for growth and long term survival of inoculants. Consider mixtures of chars Pretreatment of char to obtain desired granule size, pH, moisture conditions prior to inoculation. Incorporation with composts, rock phosphate, other additives. Application of quality control, quality assurance criteria for biochar-inoculant products.Properties of Biochar as an Inoculum CarrierLarge internal surface area provides protected habitat 2 20 uM pore spaceFor growth of bacteria and fungi in internal spacesProduction process leads to pre-sterilized medium Adsorbs nutrients and growth factorsInoculants and Nitrogen Fixation of Legumes in Vietnam, Herridge 2002Importance of Storage Temperature, and Pre-sterilization of Inoculum CarrierStimulation of plant growth by volatile gases (butane diol). Proc Nat Acad Sci 200541[PDF] Bring Biochar to the Market - 2009 Bioeconomy,Stephen.pdfBring Biochar to the Market - 2009 Bioeconomy ConferenceTerra Preta Preserved ForestPlanctomyceteVerrucomicrobiaAcidobacteriumbeta-ProActinobacteriaVerrucomicrobiaVerrucomicrobiaActinobacteriaalpha-ProProteobacteriaCFBgamma-Probeta-Progamma-ProChloroflexiNitrospiraBacillus ProteobacteriaChloroflexiDelta-Pro0.05 AcidobacteriumAcidobacteriumalpha-ProActinobacteriaAcidobacteriumProteobacteriaActinobacteriaAcidobacteriumHigh Resolution Analysis of Bacterial Species in Amazon Forest SoilsBy Oligonucleotide Fingerprinting of 16S rDNA Clones agent: Pseudomonas putidaonce per year manual applicationonce per week Bioject field fermenterwater controlPesticide control: RidomilInoculum densityAnnual application trtmt108 cfu/ml, 100 ml/ treeBioject 107 cfu/ml 20 liters/treesufficient for 50 hectaresTreatments:Steddom et. al. Phytopath. 20022011 Ecological Solutions Bioject System IIFeatures:2000 liter dual fermentation units, sequential operationSystem delivers 107 cfu/ml irrigation water for 100 hectaresInoculum produced in sterile, hot-water disposable bagsLow cost, no contamination issues, no wasteComputer operated selection of inocula for selected functionsPGPR for plant growth promotionSpecific inocula for biocontrol of different diseases and pestsFermentation system housed in mobile trailerRemote control and monitoring of system operations Remote monitoring of security using video camerasPhytophthora populations suppressed by 84% in 1998Biocontrol equivalent to chemical control for 1998 and 1999Bacteria delivered to 75 cm depthResults of field trials for control of Phytophthora root rot using the Bioject System


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