Lantana charcoal as potent carrier material for Azotobacter chroococcum

Rhizobacterium A. chroococcum (NCIM No. 5576) was procured from National Collection of Industrial Micro-organism (NCIM), National Chemical laboratory, Pune, India. The culture was maintained on Jensen medium at 30°C for further study.

The nitrogen fixation ability of bacteria was tested by applying the semimicro-Kjeldahl method (Bremner, 1965). The nitrogen fixation efficiency (NFE) was determined in terms of total nitrogen fixed per milligram sucrose consumption.

The phosphate-solubilizing efficiency was examined by the method of Pikovskaya (1948) and by determining the phosphate-solubilizing index (PSI) (Premono et al., 1996) according to the following formula: PSI=colony diameter+halo zone diametercolony diameter {\rm{PSI}} = {{{\rm{colony}}\,{\rm{diameter} + \rm{halo}}\,{\rm{zone}}\,{\rm{diameter}}} \over {{\rm{colony}}\,{\rm{diameter}}}}

The siderophore-producing ability was estimated according to the universal chrome azurol sulfonate (CAS) assay method (Schwyn and Neilands, 1987). Briefly, CAS-hexadecyltrimethyl ammonium bromide (CAS-HDTMA) solution was prepared by mixing 120 ml of CAS solution (0.121 g of CAS dissolved in100 ml of distilled water with the addition of 20 ml of 1 mM FeCl₃.6H₂O solution) and 29 ml of HDTMA solution (0.729 g of HDTMA dissolved in 400 ml of distilled water). Then, CAS agar was prepared by adding 100 ml of CAS-HDTMA solution slowly to 900 ml of sterilized Jensen medium. The spot-inoculated bacterial colonies on CAS agar plate showing orange to red haloes were considered siderophore-positive ones.

Quantitative estimation of siderophores was carried out by the method described earlier (Pyne, 1994), and percent siderophore units (PSU) were calculated using the following formula: PSU=(Ar−As)Ar×100 {\rm{PSU}} = {{\left( {{{\rm{A}}_{\rm{r}}} - {{\rm{A}}_{\rm{s}}}} \right)} \over {{{\rm{A}}_{\rm{r}}}}} \times 100 where A_r = absorbance of reference at 630 nm (CAS reagent) and A_s = absorbance of sample at 630 nm.

Multiplication of the bacterium was carried out in Jensen (nitrogen-free) medium containing (g/l): sucrose 20.0, dipotassium phosphate 1.0, magnesium sulfate 0.50, sodium chloride 0.50, ferrous sulfate 0.10, sodium molybdate 0.005, calcium carbonate 2.0, and agar 20.0; pH 7.2 (Jensen, 1951) in ambient orbital incubator shaker at 150 rpm. Flasks containing 250 ml of the medium were inoculated with 2.5 ml of active mother culture of A. chroococcum and incubated for 4–5 d at 30°C. Broth with about 109 colony forming unit (CFU) per ml thus obtained was used to inoculate the carrier material.

L. camara biomass was obtained from locally available dried wild plants. Exactly 30 kg of sun-dried biomass (stems and branches) of L. camara was used to obtain LC. It was anaerobically processed in a 300-kg pyrolysis kiln developed by the Central Institute of Agricultural Engineering (CIAE), Bhopal to obtain the LC. The successive steps were as follows:

L. camara biomass (stems and branches) was collected.

Other carrier materials were obtained from different sources as follows: wood charcoal (WC) (locally available), lignite (S & S Biotech, Nagpur, India), vermicompost (VC) (SonaVermicompost, Mandideep, India), and farmyard manure (FYM) (farm of MPVS, Bhopal, India). The carrier materials were analyzed for their chemical properties including total nitrogen estimation by the semi-Kjeldahl method of Bremner (1965) and pH and electrical conductivity (EC) (S/m) by the standard methods of Peech (1965).

All the carrier materials were air-dried, ground, and passed through a 100-meshscreen. Their pH was adjusted to neutrality with calcium carbonate solution and they were sterilized by autoclaving at 121°C for 15 min. Log phase culture of bacteria (CFU:10⁸–10⁹ per ml) was mixed with the carrier materials according to their water-holding capacity in aseptic condition (Table 1). Carriers containing culture were filled in sterile polyethylene bags leaving one-third space as void, sealed immediately, and stored at 4°C. Each treatment was carried out in two sets of three each (triplicates), one set for monthly viability assay and another set for assaying viability in 6months. From the first set, 1 g of sample was taken out at 1-month intervals, while the last sample was taken out from the second set after 6 months, so that the impact of perturbation that the first set suffered during sampling could be assessed.

Samples were taken out aseptically at 1-month intervals for the calculation of viability of inoculated bacteria. One gram of sample for each treatment was mixed in 9 ml of sterile distilled water and shaken at 120 rpm for 1 min to facilitate separation of bacteria from the carrier. The bacterial suspension was serially diluted to achieve 10-fold dilution (10−10). Aseptically withdrawn aliquot of 100 μl from each 10−6 dilution was spread onto nitrogen-free agar medium with three replicates. All plates were incubated at 30°C for 2 d; after that, the colonies formed were counted and the results were expressed in CFU per gram.

The water-holding capacity of carriers was measured for 10 g of a selected carrier according to the method applied for soil (Sumner, 1999). For the analysis of moisture retention attribute, 40 g of carrier materials pre-saturated with water was divided into four parts of 10 g each. The first part was kept in a hot air oven for 6 h at 160°C, then cooled down at room temperature for an hour and weighed to find the moisture content (x₁). The other three parts were kept for 6 months (at 4°C) and then oven-dried, cooled, and weighed (x₂) as previously. Moisture (water) held (retention) was derived by subtracting x₁−x₂ and the data were then used to obtain the values in percentage.

Culture holding coefficient (CHO), that is, CFU per gram on 0 d/water-holding capacity, was calculated for each carrier material by the formula: CHO=CFUi(x1−x)×100 {\rm{CHO}} = {{{\rm{CF}}{{\rm{U}}^{\rm{i}}}} \over {\left( {{{\rm{x}}_1} - {\rm{x}}} \right) \times 100}} where CFUⁱ is CFU in x g of the carrier material and x₁ is the weight of x g of the carrier material saturated with water.

The experiments were conducted in triplicate and three times. Statistical data were analyzed using analysis of variance (ANOVA; Tukey's multiple comparisons test) in GraphPad Prism 9 software. Standard errors were determined for all mean values, and differences at the p ≤ 0.05 level were considered significant. Post-hoc least significant differences (LSD) test was also conducted at 5% (p ≤ 0.05) probability level (Williams and Abdi, 2010).

Thirty kilograms of the sun-dried L. camara biomass yielded 9.4 ± 1.07 kg of charcoal, showing carbonization efficiency (%) of 31.33 ± 1.07. The yield and efficiency were similar to an earlier finding (Abebe, 2017).

LC showed lower N2, lower inherent moisture content, higher carbon content, and nearly neutral pH, as compared to other selected carriers (Table 2). In order to improve and promote biofertilizer technology, quality control initiatives with certain minimum specifications for marketable biofertilizers have been introduced. Thus, carrier materials used to carry the biofertilizer should have higher carbon content and neutral pH (Yadav and Chandra, 2014); both of these are shown by LC.

Water absorbed and held by LC was the highest, followed by WC, lignite, VC, and FYM (Table 3). The order of water retention percentage of carriers was FYM < lignite < VC < LC < WC. The water-holding capacity showed an inverse relation with the bulk density of the carrier materials. Thus, LC with the lowest bulk density showed the highest water-holding capacity. The same reason seems to hold in the case of the moisture loss process, as LC and WC also exhibited the lowest moisture retention attribute. LC showed the lowest water retention attribute, yet it showed a higher total amount of moisture held in it at the end of the storage period. The higher water-holding capacity seems to nullify the negative effect of low retention attribute. The highest water-holding capacity of LC enables it to take up and carry a higher volume of inoculum.

All the selected carrier materials were sterilized at the outset and found uncontaminated during 6 months of the incubation period. In as a similar experiment reported earlier, Rhizobium spp.-inoculated carrier (peat) showed no contamination after 6 months of incubation (Somasegaran and Hallyday, 1982). Of the selected carrier materials studied to determine their impact on the viability of bacteria after 6 months of inoculation, LC was the most effective one maintaining the cell density at a higher level (Table 4). Of course, the initial higher viable count of LC was due to its highest water-holding capacity. In the first month, bacterial density was found to increase invariably in all the carrier materials possibly due to the presence of residual nutrients. The population of bacterium then started declining in all the carriers. The decline was, however, the least in LC. Thus, an optimum cell viable count (8.33 × 10⁷ CFU) was obtained at the end of the storage period (Table 4). The impact of PGPR inoculation on plant growth and productivity depends a lot on the number of bacteria introduced into the soil (Duquenne, 1999).

Therefore, higher bacterial survivability in the carrier material is important to achieve sufficient bacterial density in the soil following application (Feng et al., 2002; Arora et al., 2014). The highest organic carbon (OC) and moisture content of LC as compared to the other carrier materials (Table 2) seem to help the bacterial cells to remain viable during storage.

CHO was determined for all the carrier materials to examine the contribution of their material quality to the effectivity as carrier materials and to nullify the exaggeration effect of bulk density. CHO of lignite was found to be the best one and that of LC was only better than WC (Table 5), indicating a dual trend. While in general, bulk density (lignite > VC ≈ FYM > WC ≈ LC) has an inverse relation with CHO (WC > LC > VC > FYM > lignite), material characteristics do play some role. Thus, FYM and VC or LC and WC have almost similar bulk densities, but exhibit different CHOs. LC with lower bulk density and higher water-holding capacity has, in addition, suitable material characteristics, proving it a better carrier material.

There are certain standards set for the carrier materials with respect to their physico-chemical properties and performance (Table 6). Physico-chemical data as well as results obtained from the experiment regarding LC's ability to support inoculated A. chroococcum were compared with the standards; LC fulfilled these requirements, and thus proved as a potential carrier for A. chroococcum and possibly for other microbes as well.

An ideal carrier material should be easily available, low cost, non-toxic, sterilizable, mixable, and packageable. LC got through these tests. Additionally, a carrier should have readily adjustable pH, high OC content and water-holding capacity, and the ability to allow gas exchange, particularly oxygen (Somasegaran, 1994; Stephens and Rask, 2000; Rebah, 2002; Ferreira, 2005). LC, as shown above, is not inferior to the selected carrier materials (Table 6). Moreover, the carrier must also exhibit characteristics such as tolerance to harsh environmental conditions, eco-friendliness, cost-effectiveness, and solubility in water, so that bacteria can be released easily (FAO, 1993). LC displays many of these attributes, and thus has the potential to serve as a carrier for A. chroococcum and possibly other PGPR.

Selected plant growth-promoting activities of A. chroococcum 5576 before [performance (i)] and after 6 months of the storage period [performance (f)] are shown in Table 7. The bacterium recovered from the carrier exhibited almost the same level of activity for each attribute (NFE, PSI, or PSU) as before storage. This indicates that LC as a carrier does not contain any chemical that is inhibitory to the bacterium and can permit microbes to survive during storage to the maximum extent.