ESEM micrographs of UW5-inoculated biochar depict many regions comprised of globular matrices with refraction indices that differed from biochar surfaces . These regions were typical of all inoculated biochar particles and were not on non-inoculated biochar surfaces . Additionally, igures 2.1 c-d show images indicating the presence of viable GFP expressing cells adhering to the biochar. This provides visual confirmation of inoculation specific to live UW5-pSMC21 cells. It should be noted that the Heijnen and van Veen method, described for soil cell extractions for enumeration of UW5 CFUs, was employed using inoculated biochar only. This was an attempt to enumerate UW5 cells initially adhered to the boichar. However, when applied to the biochar alone, this method never yielded reproducable results and therefore was not useful for this purpose. The soil DNA extracts obtained using the CPI protocol were all considered to be of high purity and concentrations ranged between 10 and 30 ng µl-1 . DNA purity and yield did not correlate with the presence or absence of biochar in the soil. This was an improvement over the PowerSoil®-provided protocol,strawberry gutter system which yielded 2– 7 ng µl -1 DNA with 260/280 ratios between 1.40 and 2.70 and 260/230 ratos between 0.53 and 1.85. Both the culture-based and molecular methods used to monitor UW5 cell densities in non-sterile and sterile soils yielded similar results for the proportion of initial inoculum that survived after the cells were introduced into soil.

Quantitative PCR efficiencies were 96-97% for the GFP primer sets and 105% for the 16S rRNA gene primer set. The R2 values for calibration curves were all above 0.98. The GFP copy numbers determined from microcosms prepared using non-sterilized soils were not significantly different at any time point . However, at week 3 the UW5 population densities were significantly different in non-sterilized soils with and without biochar with respect to log10 CFU counts . Specifically, microcosms prepared with previously-inoculated biochar had significantly greater CFU counts than those in which cells were directly added to soil . Regardless of the presence of biochar, UW5 inoculated into previously sterilized soils persisted at greater population densities as compared to cells added to non-sterile soils at 1 and 3 weeks after inoculation . Quantification of total bacteria present in the non-sterile soils, as measured by16S rRNA gene copy numbers, revealed that the population sizes stayed relatively constant and were not significantly affected by the presence of biochar .In all of the treatments, UW5 was present on the surface of 1-week-old cucumber roots at high population densities that ranged between 2.37×105 –1.44×106 cfu g-1 root fresh wt. . Root colonization by UW5 was not significantly different in soils amended with biochar prior to inoculation or when inoculated into the soil using biochar as a carrier . The 1% biochar application lead to significant increases in root dry weights, total root lengths, plant heights, leaf counts, and plant dry weights and also decreased soil bulk density by 17% and increased soil water holding capacity by 4%. The addition of UW5 as a cell suspension or on the biochar carrier did not result in any significant increases in plant growth parameters.

It should be noted that root branching was determined by dividing the number of forks output from WinRhizo by the total root length. As previously reported, these estimations can yield high values because overlapping roots can be counted as root forks . Hence, these values serve for internal comparison between scans of roots with similar densities. Plant root growth was not significantly different in the non-biochar-amended treatments, irrespective of inoculation with strain UW5 . GFP served as an excellent molecular and observable marker. Population dynamics were studied using both molecular methods and culture-based methods and the results concurred. The population size estimates determined from each method were approximately 3 orders of magnitude apart, but this was expected. The molecular method quantified total cells, including those that have diminished in vigor to become nonviable, whereas the culture-based method quantified only cells that are metabolically active and cell aggregates can be counted as a single colony. Also, there are multiple copies of GFP per UW5 cell. In fact, the pSMC21 parent plasmid, pUC18, is maintained in Escherichia coli at approximately 50 copies per genome . Although biochar materials have been demonstrated to reduce soil DNA extraction efficiencies in previous research , that was not the case with our modified PowerSoil® protocol. Initial GFP copy numbers, obtained for DNA extracted immediately after UW5-pSMC21 inoculum were spiked into soils, did not differ when biochar was present . Hence, we recommend the modified DNA extraction method for use with biocharamended soils.The main advantages of the inoculation method described here were the nonabrasive preparation of the microorganisms and the simplicity of the bacterial loading step. Some of the starting population densities were slightly higher in inoculated biochar treatments .

Possibly, bacteria applied to the char may have used residual nutrients from the biochar to increase in population. However, these increases were not significant and do not affect the ability to contrast population densities at later time points. Biochar particles observed after UW5 inoculation had regions that looked similar to biofilms observed at similar magnifications using ESEM . However, the dispersal of these films over the biochar was not continuous, leaving regions of char surfaces exposed. This could be beneficial if a goal of biochar amendment is to affect soil properties, such as cation exchange capacity, or to be used as a sorbent for contaminants. Additionally, the degree to which bacteria adsorb to biochar surfaces will be influenced by biochar pyrolysis temperature, feedstock, and microbial surface properties . Many studies have demonstrated that biochar produced at temperatures near 600 °C have much higher specific surface areas and are more adsorptive than biochars made from the same feedstocks at lower pyrolysis temperatures . The shelf life of the inoculum on the biochar was not tested here and would be an important parameter to assess the efficacy of this inoculation method.While our original hypothesis speculated that biochar would improve the longterm survival of the soil inoculant, the results of this research do not indicate a profound impact of biochar on the fate of the inoculant, whether added to the soil prior to inoculation, or when used as a carrier to deliver the inoculum into the soil. Both culture and molecular-based methods depicted much greater survival rates of UW5 when incorporated into sterile soils. The sterile soils were presumed to have no bacterial, fungal, protozoan, or nematode populations, the presence of which would potentially decrease UW5 cell densities over time. Hence, these serve as controls for survival when competition and predation are non-issues. When introduced into non-sterile soils, UW5 cell numbers decreased 100 to 1000 fold after 3 wks,grow strawberry in containers regardless of the presence or absence of biochar. Low temperature biochar products commonly contain a large number of adsorbed volatile organic compounds that may hinder microbial growth . These findings indicate that the addition of 2-yr old 300°C pinewood biochar to soil microcosms was not detrimental to inoculum population densities and provided a means to assure even distribution of the inoculum throughout the soils. The population density of viable cells was more than 10-fold greater, when the cells were introduced on the biochar carrier as compared the treatment in which the cells were directly added to unamended soil . In contrast, cell survival based on GFP copy numbers showed only a slight improvement in survival when the inoculum was introduced into the soil on the biochar carrier . In this regard, earlier research by suggest that alginate encapsulation may be more effective for preventing this population decline in nonsterile soils. Biochar additions had no significant effects on the total bacterial density in nonsterile soils, as determined by quantification of total 16S rRNA genes. Hence, we ruled out the possibility that the greater UW5 population densities associated with biochar amendment were simply a factor of biochar’s stim lation of total bacterial ab ndance . In contrast, used similar soil DNA extraction techniques and 16S rRNA qPCR methods to show that addition of wheat-straw biochar to soil increased the abundance of soil bacteria.

These opposing findings suggest that different types of biochar may have varying amounts of residual labile carbon and will support microbial growth differently. Overall, the 300 °C pinewood biochar served as a delivery mechanism to evenly mix viable UW5 cells into soils but the degree to which its use resulted in increased inoculum survival should be greater if this material is to be recommended as an inoculum carrier over other common carrier materials. Likewise, other biochar preparations may prove to be superior to the low temperature pine pyrolysis material tested here based on internal porosity and surface area and charge properties. Other variations not evaluated here include methods for mixing and infiltrating the biochar with bacteria, and use of supplemental nutrients to cultivate the bacteria in the char following inoculation of the char particles.Recent work demonstrated that biochar could interfere with microbial signaling and that this hindrance was different depending on biochar type . Here we observed that after one week UW5 root colonization was not hindered even when bacteria pre-colonized biochar surfaces . This time point indicates that UW5 can efficiently colonize plant roots during early development regardless of biochar presence. Enterobacter cloacae strains 501R3 and GS1 were shown to colonize cucumber and rice roots in un-amended soils at similar population densities to those observed here . Biochar provided a significant influence on both shoot and root development, which was likely a result of increased soil porosity and water holding capacity post biochar amendment . However, cucumber plant development was not significantly impacted by UW5 inoculation . Previously, demonstrated significant increases in lateral root formation and total root length when UW5 colonized canola roots at a rate of 106 following seed treatment and development in growth pouches. Here we only observed slight increases in these parameters when seeds were germinated in the absence of UW5and root population densities were closer to 105 . Plant response to UW5 may be more significant during germination or may require higher population densities. Although this density may be too low for UW5 induced plant growth promotion, Rhizobium in soils at a rates of 103 – 105 CFU g-1 soil were shown to result in significant increases in nodulation . Hence, while the pinewood biochar–UW5 combination is not ideal, the combination of biochar with other strains of PGPR may result in more direct benefits to plant development. In future work it will be important to evaluate different types of biochar in an effort to strike a balance between adsorptive properties and porosity to best optimize inoculum survival, without hindering plant-microbe interactions caused by sorption of hormones or signal compounds on the biochar surfaces.Plant growth promoting rhizobacteria are currently being develop for use as biofertilizers to improve agronomic productivity . One of the major challenges in the development of commercial biofertilizers is assuring consistent, high population densities of the inoculum, particularly non-spore forming bacteria. Carrier materials can influence inoculum success by providing protective pore spaces and also by modifying the soil structure, perhaps making it more conducive for microbial colonization . Peat moss has traditionally served as a carrier for Rhizobia and often alternative carriers are assayed in comparison with peat . Nonetheless, the use of peat and vermiculite is limited by the expense of mining the materials and by lack of availability of the materials in regions where they are not naturally present. Hence, sustainable, widely-available materials are desirable as alternative inoculum carriers. One of the most appealing new materials that could function as an inoculum carrier is biochar, which is being advocated as a soil amendment for mitigating climate change and improving soil fertility . When used as a soil amendment, biochar has been shown to condition soils, effectively decreasing the bulk density and improving aggregate formation, soil water holding capacity, and nutrient retention . To date, the economic costs associated with biochar production, transportation, and application are major factors that limit its wide-spread use . If biochar is used as an inoculum carrier, this can potentially facilitate the development of many biotechnology products for agriculture including PGPR, plantdisease suppressive bacteria, and microorganisms that are useful for bioremediation of contaminated soils.