Towards a bacterial biofertiliser for the rehabilitation of disturbed and degraded land

Agriculture, mining, industry, and human activity disturb, degrade, and pollute pristine environments and particularly the soil environment. Excessive land exploitation slows or disrupts the soil potential, rendering it incapable of playing its role leading to land degradation. In the mining sector, specifically coal mining, rehabilitation of disturbed and degraded land involves strategies that include importing topsoil and using fossil fuel-derived fertilisers. Both practices are unsustainable. To address the unsustainability, a myco-phytoremediation technology known as Fungcoal was developed to facilitate successful revegetation of mining-disturbed and degraded land following the bioconversion of waste coal into a soil-like humic-rich substrate. To offset the dependence on chemical-based fertilisers, efforts were/are focussed on finding mutualistic and cost-effective microbial resources with plant growth-promoting (PGP) activity as a bacterial biofertiliser. This study made use of 22 isolated bacteria and the three Fungcoal coal-degrading fungi viz., Aspergillus sp. ECCN 84, Aspergillus sp. ECCN 225 and Penicillium sp. ECCN 243 as the microbial resource. Initially, characterisation of the substrate waste coal and molecular identification of the selected bacterial isolates were carried out. Physicochemical analysis of the low-rank coal (LRC) substrate revealed a pH of 3.60 with background S content equivalent to 7.13 g L-1, N at 20 mg L-1, P at 7.8 mg L-1 and K at 3.3 mg L-1. Energy-dispersive X-ray spectroscopy (EDX) analysis revealed a C and O content of 23.09 and 69.03 wt%, respectively. Metagenomic analysis of the microbial population associated with the LRC substrate showed that among the 96.32% of bacteria, 59.46 to 62.18% belonged to Bacillota (also called Firmicutes), a phylum of largely Gram-positive bacteria, and 33.01 to 35.74% to Pseudomonadota (synonymous with Proteobacteria), a phylum of mostly Gram-negative bacteria. Following purification of the selected bacterial isolates and molecular characterisation by PCR, phylogenetic relatedness to known plant growth-promoting bacteria (PGPB) contained in the GenBank database showed that these bacterial isolates clustered with high bootstrap values to the reference PGPB strains. Only Pseudomonas sp. ECCN 10b (MW672582) was outside of the tree and shared significant similarity (100%) with Pseudomonas fluorescens (CP015638). A biochemical study revealed that the two Proteus sp. strains, Exiguobacterium sp., Enterobacter sp., and Ancylobacter, tolerated high salt and a wide range of temperatures. Bacterial isolates showed a high pH tolerance between 3 and 11, with the best growth at pH around 7. Nine of the identified strains, four Bacillus sp., Exiguobacterium sp., Enterobacter sp., Pseudomonas sp., Arthrobacter sp., and Aeromonas sp., were able to grow and increase in a medium containing either glucose, mannitol, sodium L-glutamate, sucrose, or fructose. Growth was highest in media containing either sodium L-glutamate, sucrose, or fructose. All the coal degrading strains and 83% of those isolated from municipal wastewater used more complex carbon sources such as high and LRC. The potential for PGP activity was quantified spectrophotometrically by measuring the production of auxins, as indole-3-acetic acid (IAA) equivalents; gibberellins, as gibberellic acid (GA3) equivalents, along with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore activity. Additionally, nutrient mobilisation was evaluated by monitoring an ability to mineralise NH4+, PO43−, and K+. Competent PGP strains for the coal degrading isolates included Proteus strain ECCN 20b, Proteus strain ECCN 23b, and Serratia strain ECCN 24b. In response to L-trp supplementation, the concentration of indolic compounds (measured as indole-3-acetic acid) increased. Production of ammonium and solubilisation of insoluble P by these strains was also apparent. Only Serratia strain ECCN 24b could solubilise insoluble K. Production of indoles increased following exposure to increasing aliquots of LRC, suggesting no negative effect of this material on indole production and that these bacteria may possess PGP potential. Of the twelve bacterial strains isolated from wastewater MaB-flocs, three produced indoles, nine mineralised NH4+, seven solubilised P, and one K. Potential of isolated strains for PGP activity according to a one-way ANOVA on ranks was: ECCN 7b > ECCN 4b > ECCN 6b > ECCN 3b = ECCN 10b > ECCN 1b = ECCN 5b > ECCN 8b > ECCN 2b > ECCN 12b > ECCN 9b = ECCN 11b. Further study revealed that cell-free filtrate from indole-producing cultures of Aeromonas strain ECCN 4b, Enterobacter strain ECCN 7b, and Arthrobacter strain ECCN 6b promoted mung bean adventitious root formation. Based on a biochemical study and the outcome of the ranking of bacterial strains according to PGP-like activities, three bacteria, Enterobacter sp., strain ECCN 7b, Proteus sp., strain ECCN 20b and Serratia sp., strain ECCN 24b that showed great mutualistic relationship with the most effective Fungcoal biocatalyst, A. fischeri ECCN 84, were used to prepare a bacterial bio-fertiliser. This consortium grew well in NB supplemented with L-tryptophan and produced indole compounds that could activate the adventitious rooting of mung bean (Vigna radiata L.) hypocotyls. Finally, the consortium showed no antibiotic resistance activity; however, they produced better biofertiliser with good responses to root/plant biomass production of the same Fabaceae, mung bean (Vigna radiata L.). The further development of this consortium into a cost-effective, environmentally friendly biofertiliser may help reduce dependence on chemical-based fertilisers and improve the sustainability of Fungcoal and other land rehabilitation strategies. Further studies are therefore underway to investigate in greater detail the PGP activity of these isolates individually and in consortium under field conditions to support the Fungcoal myco-phytoremediation strategy.