While biochar can be produced from a variety of feedstocks, the physical and chemical properties of biochar will vary depending on the type of feedstock and the pyrolysis process used to produce the material . Pyrolysis temperature refers to the highest treatment temperature achieved during the pyrolysis process and can range between 200 and 1000 °C . Additionally, various biochars show divergent effects on soil microbial activity, transport, and diversity, likely caused by indirect changes to the soil’s chemical properties . While not yet well investigated, both the feedstock and HTT will likely affect the suitability of biochars as carrier materials. The objectives of this study were to compare biochar materials to standard carriers with respect to promoted inoculum survival. Improved survival was related to physico-chemical properties of the biochar materials. Ideal physico-chemical properties were recognized and related to either feedstock or pyrolysis temperature. Overall, specific biochar feedstocks and production methods are identified for optimizing biocharinocula formulations.Carbon and nitrogen analysis was performed on a FlashEA 1112 Elemental Analyzer . Permanganate oxidizable carbon was determined using the method described by . Biochar pH and electrical conductivity measurements were determined using previously described methods . Briefly, 1 g of biochar was suspended in 20 mL deionized water and left shaking at 180 rpm for 1.5 h. The pH was measured using an Accumet® basic AB15 pH meter and electrical conductivity readings were determined using an Accumet® model 20 pH/conductivity meter . Biochar surface hydrophobicity was determined for dry, fresh biochar sieved through a 0.5 mm mesh using the molarity of an ethanol drop test . The MED values from 1– 2 indicate hydrophilic samples, 3– 4 are slightly to moderately hydrophobic, and 5-7 are strongly to extremely hydrophobic. As recommended by the International Biochar Initiative ,hydroponic nft channel specific surface areas were determined using the Brunauer, Emmett, and Teller N2 method on an ASAP 2020 Physisorption Analyzer as outlined in the Active Standard ASTM D6556 .

The percent water holding capacity for the carriers were determined after the materials were saturated in water for 24 hours, then allowed to air dry for 1 hr. Values for %WHC were calculated using the mass of water retained in the material per g dry material x 100. The physical structure and surface pore-opening diameters for the 300°C biochars were visualized using a Hitachi TM 1000 tabletop environmental scanning electron microscope . Pore-opening diameters were measured using TM-1000 software . Enterobacter cloacae UW5 was generously provided by Dr. Cheryl Patten . Microbial cultures were grown at 30°C, shaking at 170 r min-1 , in Luria-Bertani medium , unless otherwise specified. Electrocompetent UW5 cells were prepared using methods described by . UW5 cells were transformed with a rhizosphere stable plasmid, pSMC21, carrying a bright mutant of green fluorescent protein , provided here by Dr. Yanbin Guo. Transformation was carried out using 500 ng plasmid combined with100- 200 µL of competent cells, electroporated at 2.5 kV, 25µ , 250Ω, sing a 2 mm gap c vette in a Biorad GenePulser . Integration of plasmids was verified by selection on kanamycin medium and by microscopic observation of GFP expressing cells. Fluorescent microscopy visualization was performed on an Olympus IX71fluorescent microscope scope using a light excitation range 533– 583 nm, with an emission range of 607– 684 nm. The quantity of indole compounds produced by transformed cells was compared to that of wild type UW5 using Salkowski reagent and the S2/1 method described by . The UW5-pSMC21 transformants were screened for growth inhibition using growth curve analysis on a nutrient rich LB media and a carbon and nitrogen starvation response media prepared according to . The stability of plasmid pSMC21 in strain UW5 was assayed over a 2 week period. Cells were transferred daily to fresh Voigt media without kanamycin and at 3 d intervals cultures were serially diluted and spread onto plates with and without kanamycin. The percent of cells retaining plasmids was calculated based on differences in CFU counts on these plates.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 treatments. To prepare the liquid inoculum, 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. This constituted the liquid inoculum, final cell density of 5.6 X 109 ± 0.3 CFU ml-1 , that was used for all treatments. Twenty milliliters of liquid inoculum were left shaking at 25 °C for 24 h with 2 g of carrier material in 125 ml flasks. Treatments were prepared by thoroughly mixing inoculated carriers with 20 g soil or by mixing 20 ml liquid inoculum directly into soil, providing a final carrier application rate of 1% . Four replicate microcosms were prepared for each treatment soil in 200 ml plastic cups with drainage holes and foam tops to allow water and air flow. DNA was extracted from each replicate after the initial inoculation. Microcosms were weighed daily and watered with deionized water to maintain microcosms at 60% field capacity. After 4 weeks a second round of DNA extractions were performed on all replicate microcosms. The soil DNA extractions served as templates for qPCR used to quantify GFP gene copy numbers.DNA was extracted from 0.25 g of soil using the PowerSoil® DNA isolation kit from MoBio Laboratories with modifications . All extractions were tested for purity and concentration using a NanoDrop 1000 . All qPCR reagents, protocols, and data analysis were performed within the standards outline by the MIQE guidelines . Reactions were set up using the SsoAdvanced universal SYBR® Green supermix and were run on a MyiQ® Thermal Cycler . For the survival study, GFP primers and qPCR cycle conditions and melt curve analyses were identical to those described in Chapter 2. All qPCR reactions involving sample DNA or control DNA templates were prepared in duplicate. All of the biochar materials tested here were shown to be useful as inoculum carriers for the PGPR strain E. cloacae UW5, but also varied in their efficacy. This appeared to be based on differences in the chemical and physical properties of the individual biochars. Among the materials, Pine600 was identified as the best biochar for use as an inoculum carrier. It performed as well as the industry standard carrier, peat moss,nft growing system and its use resulted in higher sustained population densities than did vermiculite. All biochars tested performed as well as vermiculite and none demonstrated detrimental effects on the UW5 population. Peat moss supported the highest cell density in samples analyzed after the 24 h inoculation procedure and also promoted the greatest survival after the 4-week soil incubation, which was slightly higher than that of the Pine600.

This was associated with the high availability of labile carbon and high nitrogen content of the peat . To identify specific characteristics that related to the survival outcomes, the biochars were assessed based on several chemical and physical parameters. All characteristics analyzed were highly variable among the various biochar materials, which is consistent with previously reported findings . The pyrolysis temperature had the greatest effects on pH and SSA, whereas feedstock type largely determined the % WHC of the biochars. Biochar pH and C:N ratio had the greatest effect on initial GFP copy numbers, which reflect the direct effect of the carrier on the inoculum during preparation. The population density was fit to pH via a Gaussian distribution, which identified an optimal pH range for biochar as an inoculum carrier for the test strain. After inoculation, the Pine300 biochar, which had a pH of 4.63, the lowest of the biochars, also supported the lowest starting cell density . However, after 4 weeks in the soil, this material supported cell densities that were similar to those supported by the other biochars and vermiculite. Also, when cell densities were compared after the 4-week incubation, there was no correlation between the biochar pH and survival. Hence, while the pH may have been initially influential, after application to the soil, this effect was no longer detected. Other variables associated with higher initial population densities were related to nitrogen in the char, lower C:N ratios and higher N contents. Saranya et al.also observed a positive influence of nitrogen when testing the shelf life of Azospriliium lipoferum soil inoculants on various biochars. However, in the present study, there was no relationship between biochar nitrogen contents and cell densities after 4 weeks of incubation. We also noted that the top performing carriers, Pine600 and peat, were moderately to strongly hydrophobic when tested as a dry materials, yet they have high % WHC’s. The hydrophobicity was assayed on dry materials, but % WHC values were obtained after 24 h of saturation. Hence, the hydrophobicity of the dry biochar does not appear to be a key concern when evaluating the utility of biochar as an inoculum carrier. This also indicates the importance of sufficient inoculation periods to ensure infiltration of the material if using liquid inoculum. Survival of the introduced PGPR strain UW5 after 4 weeks in non-sterile soil was strongly correlated with the C:N ratios of the biochar materials. Soil C:N ratios can influence soil microbial community composition and in particular have shown positivecorrelations with total phospholipid fatty acids . In agreement with this finding, a recent study demonstrated a positive relationship between the C:N ratio of biochar-amended soils and soil total PL A’s and bacterial PL A’s, in particular . However, Jindo et al.report a negative correlation between C:N ratio and bacterial biomass in biochar-compost mixtures. Altogether these findings indicate that biochar application will influence soil C:N ratios, and that C:N ratio will have an important effect on soil bacteria, but that this effect may be inconsistent across variable soil types. Several other parameters were related to week 4 survival when fit to Gaussian models. In particular, biochars having SSA’s, pore-opening diameters, and % WHC’s in the mid ranges maintained greater UW5 population sizes.

These physical characteristics depend on the surface structure of the biochar materials. Two of the biochars, Pit600 and Shell600, had the highest SSA’s b t did not res lt in improved inoculum survival. Previous research demonstrated that biochars prepared from the same feedstocks had increasing microporosity and SSA’s with increasing final HTT’s . These materials may have a large volume of nano– micropores, which are not accessible to bacteria and thus do not reflect the functional carrier capacity of the material. In fact, macroporosity often makes up only a small portion of the total surface area on biochar particles . The pore-opening diameters will determine which fauna are excluded from the biochar interior pore space and whether they are accessible to bacterial inoculants. Here we only visualized the pore-openings of the 300°C biochars, based on the ass mption that the higher HTT’s will have a significant effect on micro to nanoporosity which was measured by SSA, not the macropores we visualized using ESEM. The materials closely resemble that of the feedstock at a cellular level, as has been reported previously . The biochars with pore-opening diameters between 26 and 46 µm were ideal. Pores in this size range could play a significant role in protecting pre-established colonies from predation. Overall, pretreatment of chars can change some of their chemical properties but, unless blocked, pore-openings are not easily distorted. Thus, the physical properties and surface features of a potential feedstock should be an important consideration when selecting a biocharbased carrier. The effect of PGPR on native microbial communities will significantly impact its utility as an agricultural soil amendment. To assess the bio-availability of residual phosphorus in biosolid-derived biochars it is important to consider the mineral phosphate solubilizing microbial community. In soils phosphate is frequently complexed by calcium , iron , or aluminum , making it insoluble and unavailable to plants . This is also the case with the residual P in biosolid-derived biochar studied here, where P is predominantly complexed as Al and possibly Ca phosphates .