All together, it is clear that with biochar addition to a soil there will likely be changes in soil biological community structures and functions and that pyrolysis temperature, feedstock, and application rate will all have significant influence on these induced changes. Pre-inoculation of biochar with PGPR could add value for biochar marketing and provides a means to evenly distribute and potentially improve the survival of inoculants following their introduction into soils. Many biochars have characteristics that also are conducive for use as inoculum carriers including high internal porosity, large specific surface area, and the ability to adsorb organic compounds and bacteria . As apparent in Figure 1.7, biochar materials, specifically those prepared from lignocellulosic feedstocks, are covered in pores averaging about 20 µm in diameter. Pores of these sizes are considered to be accessible to bacteria, but will likely exclude entry of bacterial predators such as nematodes and protozoa. Biochar is sterilized during pyrolysis, which can better ensure high quality preparations of carrier materials . Establishment of a plant-beneficial bacterial population on biochar also provides preemptive colonization of the biochar substrate and may help to competitively exclude colonization by plant pathogens .The PGPR activity of a strain in the introduced soil environment is a requirement for an effective biological fertilizer. There is concern that bacterial enzymes or substrates could adsorb to char surfaces or be regulated by small molecules incorporated with char. Biochar had an effect on plant gene regulation and was shown to interfere with microbial signaling.
Thus, it is essential to ensure that biochar does not interfere with plant-microbe signaling essential for root colonization and that the PGPR activity of a strain of interest is not impeded in the presence of biochar. Current methodology employed to study microbial presence and activity in soil is dependent upon the extraction of whole cells, enzymes,planting gutter or nucleic acids from soil. For instance, many methods for analyzing soil microbial diversity and microbial activity are reliant upon on extraction of highly-pure DNA directly from soils Dobrovol’skaya et al., 2001). However, DNA is negatively charged due to the phosphate ions in its structure and could adsorb to newly-made biochar particles. As biochars weather, they develop a dominant negative charge and thus may interact with DNA in other ways such as a cation bridging type of bonding . Furthermore, macropores may shelter bacterial colonies during extractions and whole cells or DNA from these microorganisms will be omitted from further analysis. Furthermore, components of soil organic matter can contaminate extracted DNA with inhibitors of polymerase chain reaction -based downstream applications. Humic acids, which are very common in soils, are renowned for interfering with reagents used in DNA extraction kits. Hindrance of extraction efficiency has also previously been demonstrated in soils with heavy clays . The large-scale production of biochar for carbon sequestration provides an opportunity for using these materials as inoculum carriers to deliver plant growthpromoting rhizobacteria into agricultural soils. Biochar could serve as a cost effective, widely-available carrier. Previously, an Azospirillum biofertilizer was demonstrated to have a shelf life of at least 6 months at room temperature when carried on biochar . However, little is known about the outcome of the inoculum if introduced to soils. Whether biochar actually can promote survival of bacterial inoculants in soil is a topic that has not been rigorously investigated.
The gene encoding a green fluorescent protein was originally isolated from the jellyfish, Aequorea victoria, and is unlikely to be present naturally in agricultural soils . Hence, it serves as an excellent molecular and observable marker and has successfully been used to monitor bacteria added to non-sterile environments . Both molecular and culture-based methods have shortcomings when used to assay soil bacteria . The combination of these approaches, using quantitative PCR and colony forming unit counts to simultaneously track GFP-tagged UW5 offers a reliable picture of inoculum population densities over time. If biochar stimulates microbial growth in soils, quantitative data on the UW5 population size may reflect this consequence and not necessarily improved inoculum survival rates. The response of soil bacterial population size to biochar amendment was also monitored using universal 16S DNA primers. An ideal carrier will not only prolong inoculum survival but also will not interfere with processes essential for microbial induced plant growth promotion. These experiments also examined differences in root colonization and assayed cucumber root and shoot development following different methods of soil inoculation. The objectives of this study were to optimize several methods that can be applied to samples of biochar-amended soils. Here we examine the effects of a pinewood biochar that was produced with a highest treatment temperature achieved during pyrolysis of 300 °C. Cell densities of E. cloacae UW5 were enumerated in soil after incorporation into soil already containing biochar or when added to soil using biochar as an inoculum carrier. Seed treatment with strain UW5 has been shown to increase total root length and lateral root branching in mung bean and canola which was correlated to exogenous production of indole-3-acetic acid . The phytohormone, IAA, result in stimulation of plant root growth and disease resistance when produced by PGPR . Strain UW5 serves as a well-studied bacterium that has an understood, tryptophan-dependent pathway for IAA production. We hypothesized that a pinewood biochar, with large internal porosities, could provide protected habitats for inoculum, thereby reducing predation by nematodes and protozoa and prolonging survival.
An Arlington sandy loam, collected from a field with previous agricultural history from the University of California, Riverside , was passed through a 4 mm sieve and used for all experiments. Pine-wood biochar produced at a maximum pyrolysis temperature of 300 °C was provided by Alterna Biocarbon and was applied to soil at a rate of 1% two years after its production. This biochar can be classified as a low-temperature and was characterized according to protocols outline by the International Biochar Initiative “IBI Certification Program Manual: Re irements and Proced res for Biochar Certification,” 2013. Triplicate measurements were taken using an Accumet® basic AB15 pH meter for pH and for electrical conductivity , using an Accumet® model 20 pH/conductivity meter . The biochar was visualized using a Hitachi TM 1000 tabletop environmental scanning electron microscope . Pore-opening diameters were measured using TM-1000 software . To sterilize,gutter berries soil was autoclaved in polyethylene bags and autoclaved again after 48 h to ensure sterility. Cucumber seeds, Cucumis sativus cv. Spacesaver, were uniformly germinated by heating in 45°C deionized water for 5– 10 min. The heat-treated seeds were maintained on DI water soaked filter papers in glass Petri dishes in the dark for 2– 3 d before planting. Germinated seeds were planted in 50 cm3plastic cones for samples collected at 3 week or less, or in 1700 cm3 pots for plants grown for more than 3 weeks. Seeds were covered with 1– 2 cm soil, and the plants were maintained in growth chambers at 25 °C, with 50% humidity, under 12 h photoperiods provided by fluorescent and incandescent light. One week after plant emergence, pots were mulched to decrease soil drying. Plants were fertilized biweekly with half strength complete nutrient solution and watered with 50 ml or 5 ml every other day in the first 3 weeks and then daily thereafter.UW5 cells were tagged with a bright mutant of green fluorescent protein carried on rhizosphere stable plasmid, pSMC21, a derivative of pSMC2 developed by . Electrocompetent UW5 cells were prepared using methods described by . Transformation was carried out using 500 ng pSMC21 combined with 200 µl of competent UW5 cells, electroporated at 2.5 kV, 25µF, 250Ω, sing a 2 mm gap c vette in a Biorad GeneP lser Herc les, CA. SOC medium was added immediately after electroporation and cultures were incubated at 28 °C for 1.5 h, after which they were spread plated onto LB agar containing kanamycin. Integration of pSMC21 was verified by sequencing of PCR products amplified from the GFP gene and by microscopic observation of GFP expressing cells.
Fluorescent microscopy was performed on an Olympus IX71fluorescent microscope scope using a light excitation range 533– 583 nm, with an emission range of 607– 684 nm. UW5-pSMC21 transformants were screened for altered growth response relative to the wild type strain by growth curve analysis on nutrient rich LB medium and a carbon and nitrogen starvation response medium prepared according to . IAA production levels by the transformed cells were compared to those of wild type UW5 using Salkowski reagent and the S2/1 method described by . The stability of plasmid pSMC21 in strain UW5 was assayed on Voigt carbon and nitrogen starvation media. Cells were transferred daily over a 2-week period, to fresh Voigt medium without kanamycin and at three day intervals cultures were serially diluted and spread onto plates with and without kanamycin. The percent of cells retaining the plasmids was calculated based on differences in CFU counts on these plates.To prepare the liquid inoculum used for all treatments, UW5-pSMC21 cultures were grown overnight to late log phase in LB + kanamycin. Cultures were washed twice with sterile 0.85% NaCl using 30 min centrifuge steps at 4000, 4°C. Washed cell pellets were brought to ½ initial culture volume with sterile 0.85% NaCl. Dilutions of this s spension were spread onto LB + kanamycin plates and C U’s were counted to determine starting inoculum concentrations for all experiments. The washed inoculum was left shaking at 25 °C for 24 h without biochar, for liquid inoculum, or in a 5:1 inoculum: biochar mixture, to be used as inoculated biochar. Direct inoculation treatments were prepared by thoroughly mixing 50 ml liquid inoculum into 1 kg soil by hand, ensuring even wetting. Inoculated biochar treatments were prepared in a similar fashion, using a 50 ml liquid, 10 g biochar mixture for each kg soil prepared. All inocula were added to soils with or without biochar carriers at a rate between 7×106 and 7×107 CFU g-1 soil. Soils receiving no inoculum were treated with 0.85% NaCl. The presence of UW5 on biochar surfaces was verified using ESEM and fluorescence microscopy. Biochar particles suspended in sterile 0.85% NaCl served as negative controls. ESEM images were obtained using a Hitachi TM 1000 tabletop microscope. To preserve microbial cells prior to imaging, biochar samples were flash-frozen in an isopentane bath chilled with liquid nitrogen. For each treatment, 10– 15 electron micrographs were collected. GFP expressing UW5-pSMC21 cells were imaged on fine biochar pieces using fluorescence microscopy and conditions previously described. For determination of UW5 population density by CFU counts, bacterial cells were extracted from soils . Briefly, 10 g of soil were added to a 250 ml flask with 95 ml of sterile 0.1% sodium pyrophosphate and small marbles . Flasks were shaken at 200 r min-1 for 10 min and allowed to settle 1 h. Using standard techniques, suspensions were serially diluted and spread onto LB agar plates supplemented with cycloheximide, ampicillin, and kanamycin. Plates were incubated at 28 °C for 20 h, then moved to 4 °C for 24 h to allow GFP development. Only colonies expressing green fluorescence under UV excitation were counted. At each collection point, 3 cell extracts were collected per treatment, 1 from each replicate microcosm. All extracts were spread in triplicate. To assess the effects of biochar and UW5 on plant development five differing treatments were prepared . Germinated cucumber seeds were planted according to conditions previously described with 5 replicates per treatment for each sampling time point. Plants were destructively harvested after 1, 3, and 5 weeks. To determine total root length and lateral root formation, roots were washed then stained with 0.1% Toludine O Blue solution for 5 min. Residual stain was removed by washing twice with DI water and shaking in DI water for 5 minutes. After staining, the roots were imaged using an Epson Expression 1680 scanner and images were processed with WinRHIZO Pro 2005a software . This program provided measurements on total root length as well as total number of forks for scanned root clusters. After imaging roots were oven dried at 80 °C for 24 h and weighed to determine dry weight biomass. At week 5, plant fresh and dry weights, total leaf counts, leaf widths, and plant heights were also measured.Plasmid pSMC21 was retained in greater than 95% of the UW5 cells after 42 generations of growth on carbon and nitrogen limited medium.