Development of Bacteria biofertilizers using locally isolated rhizosphere populations and agricultural refuse and their impacts on growth of local test crops

Abstract Biofertilizers are the preparations of live microorganisms added to the root, seed or soil to promote plant growth. In this study, Plant Growth Promoting Bacteria able to solubilize insoluble phosphate (P) and potassium (K) forms were isolated, characterized and identified. Two isolates that demonstrated excellent solubilization of potassium or phosphate from abundant and bio-available waste biomass (rice husk and cattle bone) were used to produce biofertilizers by solid-state fermentation. The biofertilizers were applied to grow three food security crops, Zea mays, Solanum lycopersicum, and Arachis hypogea, in a screenhouse, and monitored for growth impacts. Treatments A, B, and A + B biofertilizers caused a significant (p < 0.05) increase in plant dry weights. The highest microbial colonization was obtained from treatment A + B (for S. lycopersicum) with a microbial count (log 2.89 (108) cfu/g), whereas treatment with B (for A. hypogea) had the least microbial count (log 2.73 (108) cfu/g). Maximum values of experimental parameters: shoot height, leaf number, plant dry weight and leaf width were obtained with the combined application of both biofertilizers. P and K solubilizing PGPB have shown potential for use as biofertilizers in growing these key crops under the soil conditions and in the environment studied. A NOVELTY STATEMENT This work demonstrates a first, sensitive and reliable method for low-cost, sustainable, eco-friendly production and utilization of biofertilizers for improved growth of major tropical food security crops using native bacterial strains in a defined tropical agronomic environment. Its novelty is the choice of Bacillus cereus and Pseudomonas aeruginosa singly and in combination (synergy/additive) as biofertilizers for growing tropical test crops. It also demonstrated the use of a novel and cheap delivery method/carrier that ensured the establishment and persistence of PGPB in the rhizosphere. Native B. cereus and P. aeruginosa were able to solubilize and make available to plants phosphate and potassium, thereby improving soil quality and plant growth while the process achieved the reuse of waste biomass.


Introduction
The global population is estimated at over 7.7 billion and is set to grow to 9 billion in 2050, and rapid urbanization has led to greatly intensified agricultural activities for enhanced food production (Giller et al. 2021). Agricultural activities generate many by-products, including fruit bark and stems, predominantly composed of cellulose, lignin, and hemicelluloses (Zainudin et al. 2022). They become major environmental pollutants, release unpleasant smells, are connected to contaminating surface and ground waters, soil, and atmosphere, and have negative impacts on public health (Duque-Acevedo et al. 2020). Although these by-products are resource-rich, the inability to reprocess them means they persist in the environment as pollutants. Recycling of wastes reduces the unpleasant odors of garbage, benefits sanitation and public health, and can enhance soil fertility.
To improve soil fertility and enhance crop production from available arable land, farmers use various fertilizers, a significant portion of these being inorganic or mineral fertilizers. Unfortunately, only about 0.2% of applied inorganic fertilizers get used by plants, while the rest are precipitated by metals in the soil (Islam et al. 2019). To overcome these challenges and increase soil health, biofertilizer application has emerged as an attractive alternative to chemical fertilizers. Biofertilizers are the preparation of living microorganisms; when applied to seeds, roots or soil, they enhance the bio-availability and uptake of essential nutrients (Fasusi et al. 2021).
Microorganisms used as biofertilizers competitively and protectively colonize plant root systems. They become involved in the production of phytohormones, solubilization and mobilization of macronutrients such as potassium and phosphorus through chelation of cations, and mineralization by lowering rhizosphere pH by producing organic acids including malic acid, acetic acid, oxalic acid, citric acid and gluconic acids and also by the production of phosphatases (Zhang et al. 2021). They can also contribute to plant resistance to stress and pathogens through various mechanisms (Daniel et al. 2022). The use of biodegradable agro-waste in biofertilizer production is proliferating worldwide and could be one of the best ways to reprocess polluting agro-waste and improve soil fertility. This is particularly the case in significant food secure countries.
Several physiologically unrelated microorganisms, including cyanobacteria, archaea, bacteria and micro-fungi, have been used as biofertilizers to grow different crops on different soil types and agro-ecological regions, employing various agronomic practices (Fasusi et al. 2021). Bacteria, including Azospirillum, Micrococcus, Xanthomonas, Alcaligenes, Pseudomonas and Bacillus, have been produced and demonstrated to be effective as biofertilizers for use in growing a variety of crops (Glick 2020;Sun et al. 2020). Used singly and as mixed populations and consortia, they have differentially and crop-specifically achieved up to 40% improvement in crop productivity (Mitter et al. 2021).
Although biofertilizer application has gained traction in global agriculture, its use in food insecure developing countries are still facing challenges. Key among these relate to delivery systems, establishment and persistence of organisms in the rhizosphere, and efficacy of candidate organisms under given agronomic conditions, mainly when the isolate is not native to the environment of use. Currently, there are limited studies relating to their production and use in developing countries and an absence of information on the use of local candidate microorganisms as biofertilizers for improving the growth and productivity of major food security crops in typical fertility-use-challenged tropical soil types.
This study was therefore implemented to develop biofertilizers using locally isolated rhizosphere populations of bacteria (Bacillus cereus OM328186 and Pseudomonas aeruginosa OM328188) and abundant, highly polluting agricultural refuse and to apply them in the native environment in a pot culture to investigate their establishment in the rhizosphere as well as their impacts on the growth of local test crops of significant food security interest.

Collection of substrate and rhizosphere soil sample
Rice husk samples were collected from multipurpose cooperative society rice mill Abakaliki, Abakaliki Local Government Area of Ebonyi State, Nigeria (longitude 7 57 East and latitude 6 10 North) in clean plastic bags, while waste cattle bones were collected from the meat market in Abakaliki, in clean plastic bags. All samples were transported to the laboratory for immediate processing, as detailed below. In addition, rhizosphere soil (5 g) was collected by carefully uprooting young (4-week-old) S. lycopersicum, A. hypogea and Z. mays plants from depths of 5 cm to 15 cm and vigorously shaking to remove the soil loosely adherent to the roots. The plants were then collected in clean, dry, and sterile ziplock bags (Zhu et al. 2022) and taken to the laboratory for microbiological analysis.
Isolation/screening of rhizosphere bacteria for phosphate and potassium solubilization Rhizosphere bacteria from Solanum lycopersicum, Arachis hypogea, and Zea mays plants were isolated from 1 g of soil, tightly adhering to the roots of the growing plants by serially diluting the recovered soil up to 10 folds (Bhavya et al. 2017). The plates were incubated aerobically at 30 C for 24 to 48 hours. A total of 45 morphologically different colonies obtained from three replicate experiments were sub-cultured on nutrient agar plates for further purification and analysis. All 45 isolates were screened for ability to solubilize phosphate using Pikovskaya's (PVK) medium (Pikovskaya 1948), composed of (g/L) glucose, 10; yeast extract, 0.5; (NH 4 ) 2 SO 4 , 0.5; MgSO 4 .7H 2 O, 0.1; KCl, 0.2; NaCl, 0.2; FeSO 4 .7H 2 O, 0.002; MnSO 4 .7H 2 O, 0.002; Ca 3 (PO 4 ) 2 , 5; agar, 20 and pH 7. Plates were incubated aerobically for seven days at 30 C. Isolates forming clear halo zones around the colony indicated the ability to solubilize phosphate and were measured in centimeters (cm) (De Freitas et al. 1997). The solubilization index (SI) was calculated according to the method described by Sane and Mehta (2015). For potassium solubilization studies, the 45 bacteria isolates were aerobically cultured in an Aleksandrov agar medium (5.0 g Glucose, 0.5 g MgSO 4 . 7H 2 O, 0.1 g CaCO 3 , 0.006 g FeCl 3 , 2.0 g Ca 3 (PO 4 ) 2 , 3.0 g potassium aluminum silicate (as potassium source) and 20.0 g agar in one liter of deionized water) by the spot test method (Hu et al. 2006). Colonies showing clear zones were selected as potassium solubilizers and the zones were measured in centimeters (cm). Solubilization Index (SI) ¼ D/d ¼ (Diameter of zone of clearance/Diameter of growth) was determined.

Quantitative determination of phosphate (P) and potassium (K) Solubilization
The isolates were grown in the PVK liquid medium to quantify P solubilization using the Phospho-molybdate blue color method (Murphy and Riley 1962). A 100 mL of Pikovskaya (PVK) broth containing 0.5% Ca 3 (PO 4 ) 2 was inoculated with one mL of 24 hours bacteria culture (1.4 Â 10 6 cfu/mL) and incubated aerobically at 30 C on a rotary shaker (100 rpm) for 15 days. About 10 mL of each culture was collected and centrifuged at 10,000 rpm for 15 minutes to remove bacteria cells and other insoluble materials. The available P was quantified using an atomic absorption spectrometer (AAS) (6320 D model, USA) at 882 nm. To quantify K solubilization, one mL of 48 hours old bacterial culture (1.4 Â 10 6 cfu/mL) was inoculated into 100 mL Aleksandrov broth and incubated aerobically at 28 ± 2 C for ten days. The growth suspension was centrifuged at 7,000 rpm for 10 minutes and the supernatant was filtered using Whatman No. 1 filter paper. The supernatants were aspirated using a flame photometer (Jenway PEP7 Flame Photometer, Germany). The emissions were recorded and the concentrations were calculated using a potassium calibration curve (Pearson 1976) prepared using various concentrations of 10 ppm KCl solution, i.e., 0.5, 1.0, and 1.5 ppm

Molecular identification of test isolates
Extraction and sequencing of DNA The genomic DNA of the isolates was extracted using miniprep (manufactured by zymo research) (Sambrook et al. 1989). The 16S rDNA nucleotide sequence was determined by direct PCR sequencing using the BigDye terminator v3.1 cycle sequencing kit in a GeneAmp 9700 PCR System Thermal cycler. The 16S rDNA gene sequences obtained from the automatic sequencer were edited using the Bioedit6 software and identified using the basic local alignment search tool (BLAST) at >95% identity on National Center for Biotechnology Information (NCBI) GenBank database (www.blast.ncbi.nlm.nih.gov) (Kim et al. 2012). A phylogenetic tree was constructed by the neighbor-joining method using the software MEGA 4, according to Chen et al. (2006) . Sequences were deposited in the genebank and assigned accession numbers and are available on the NCBI database.
Preparation of substrate for production of biofertilizer compost To prepare biofertilizer (carrier/delivery compost), waste cattle bones collected in clean polythene ziplock bags were crushed to fine particles using a heavy-duty milling machine to obtain particle sizes ranging between 0.1 and 1.5 mm, while the rice husk was pulverized using the same machine. Rice husk was mixed thoroughly with a cattle bone in the ratio 0f 5:1 and measured into eight 2.5 L Winchester bottles (used as solid substrate fermentation bioreactors). About 500 mL of distilled water was added to each bottle containing 800 g of the husk-bone mixture before sterilizing using an autoclave at 121 C for 15 minutes.

Inoculation of substrates with candidate biofertilizers organisms
Isolates Gs-1e and Ts-3b, selected for their high ability to solubilize potassium and phosphate, respectively (being the best of the total isolates tested), were cultured in a nutrient agar plate for 48 hours at 30 C (Dagde and James 2016). Plate cultures of the isolates were washed into 10 mL of sterile nutrient broth and diluted to a population of 1.0 Â 10 8 cfu/mL each (as determined by plated count on NA). A 50 mL volume of the prepared inoculum was used to inoculate 800 g of the pretreated sterile husk-bone mixture in single (A and B) and mixed (A þ B) (25 þ 25) preparations. Two bottles containing 800 g of the pretreated rice husk and cattle bones were included as a control (uninoculated). About 1000 mL of sterile distilled water was added to each substrate to achieve a moisture content of approximately 65%. The inoculated and uninoculated bottles (in duplicate) were plugged with cotton wool, agitated manually periodically (12 hourly intervals), and incubated aerobically at 35 C for 42 days (Neto et al. 2017). After incubated (composting), the products now considered the biofertilizers and control were emptied into sterile shallow aluminum containers, covered with aluminum foil and dried by forced air at 37 C for 48 hours before being transferred into and sealed in presterilized containers and stored at 4 C until use (within one week).

Analysis of biofertilizers compost for microbial population and physicochemical parameters
The biofertilizer compost samples were analyzed at 14 daily intervals for microbial population count by standard aerobic plate count method on nutrient agar. A 1 g sample of biofertilizer compost was drawn from each fermentation bottle and serially diluted up to 10 À6 in physiological saline. The plates were incubated for 18-24 hours at 30 C and cell population as cfu/g was determined. Physico-chemical parameters include temperature using a Scanning thermometer (model Digi-Sence 69202-30 (USA)) (Hamouda 2016), pH using glass electrode (pHep Hanna (Italy)), Nitrogen content by micro-Kjeldahl method and moisture content were determined by standard methods of the AOAC (1990). Electrical conductivity was measured using an EC meter (ICM model 71150), while potassium content was measured using a flame photometer (model PFP7, Jenway, England) by Jackson's-flame photometric method and available phosphorous by atomic absorption as described by Olsen et al. (1954).

Pot experiment
The biofertilizer products were tested for their ability to enhance crop growth and yield by measuring impact in pot cultures using three different test crops (Solanum lycopersicum, Arachis hypogea, and Zea mays). 5 kg of quantities of sandy and clay soil in the ratio of 1:1 were sterilized by autoclave (121 C for 20 minutes) and placed into each of 45 sand pots. The three biofertilizer products were added (at a level of 5% of the soil weight) to each pot and mixed thoroughly. Controls were set up using the uninoculated substrate. Two healthy seeds of Zea mays, Arachis hypogea, and Solanum lycopersicum were sown separately in each pot and watered at intervals as needed for optimum growth from 2nd April 2021 to 2nd July 2021 in a screenhouse. All plant growth experiments were set up in triplicates.

Plant growth measurement
The shoot heights, leaf numbers, length, width, and plant dry weight were determined according to Indumathi (2017) at two weekly intervals for three months. The measurements obtained were compared between the three biofertilizer types (A, B, and A þ B) against the biofertilizer negative controls set up using the un-inoculated rice husk and bone (C) controls.

Re-Isolation of biofertilizer isolates from pot culture
Rhizosphere bacteria from cultivated pot plants were isolated from 1 g of soil, tightly adhering to the roots of the growing plants taken at the end of the growth experiment. The soil samples were serially diluted and plated out on Pikovskaya agar and Aleksandrov agar, respectively, as earlier described, to ensure the presence and abundance of the isolates. Purified isolates were compared with the original to determine consistent reisolation of the original biofertilizer isolate/inoculum.

Statistical analysis
The data obtained were analyzed using both one and twoway analysis of variance (ANOVA) in Statistical Product and Service Solution (SPSS) version 20.0 and Principal component analysis (PCA), which were presented as Mean ± SD. Mean values with p < 0.05 of the result were accepted as significant.

Qualitative screening of isolates for phosphate and potassium solubilization
Out of a total of 45 bacteria isolates that were examined for their ability to solubilize phosphate, seven isolates showed clear zones of solubilization with a solubilization index (SI) ranging from 2.40 to 4.60, as shown in Figure 1A. There were statistically significant (p < 0.05) differences in the solubilization indices produced by the isolates. Solubilization indices obtained with isolates Ts-3b, Ms-1c and Ts-2c were not significantly (p < 0.05) different from each other but varied from isolates Gs-2a, Ms-1d and Ms-3d, which together were comparable but differed significantly from isolate Ms-1e.
Six of the 45 isolates showed very well-defined zones of potassium solubilization on the Aleksandrov agar plate. Isolate Gs-1e and Ts-1b produced the highest solubilization index of 2.0, while isolate Ts-1c showed the least solubilization index of 1.50 ( Figure 1B). Except for Gs-1e and Ts-1b, statistical significance (p < 0.05) exists among and between the solubilization indices produced by the other isolates.

Quantitative estimation of phosphate and potassium solubilization
The quantitation of Phosphate release in Pikovskaya broth by the isolates was determined on day 15 (Figure 2A) with changes in pH of the broth cultures. Ts-3b showed the highest solubilization at 0.59 mg/L, which was associated with the most decline in pH of the culture medium to 4.3. Isolate Gs-2a achieved the least phosphate solubilization of 0.238 mg/L, which was conversely associated with a slight decline in medium pH to 6.3. A significant (p < 0.05) difference was observed between the solubilization indices when the different isolates were compared down the group. The pH showed no significant (p > 0.05) difference when Ms-1e and Ms-1d isolates were compared and no difference between Ts-2c and Ms-1c. The isolates achieved potassium solubilization that ranged from 43.67 ppm to 52.33 ppm ( Figure 2B). The result of the SI and pH showed a significant (p < 0.05) difference when the isolates were compared. Isolate Ts-1a (52.34 ± 0.01), which produced significantly (p < 0.05) higher solubilization compared to other isolates, did not cause the most decline in pH. However, isolate Ts-1b, which produced the least potassium solubilization, was also associated with the least reduction in pH of the medium. Thus, while the ability to solubilize was associated with reduced pH, the pattern was not as clear as for phosphate solubilization.

Molecular identification of bioferilizer isolates
The genomic DNA and PCR amplicons of the 6 (six) selected isolates (Ts-2c, Ts-3b, Ms-1c, Gs-1e, Ts-1a, Ts-1b) that showed the highest solubilization for phosphate and (or) potassium were obtained and estimated at 1500 bp based on the mobility of a 1 kb Hyperladder marker. Table 1 shows the bioferilizer candidate isolates' identity and their NCBI assigned accession numbers. The phylogenetic trees constructed using the Neighbour-Joining method at 1000 bootstrapping are shown in Figures 3(A-B) for the two candidates used for biofertilizer pot plant trials. Phylogenetic trees for the other isolates are available as Supplemental material.

Physicochemical parameters of biofertilizers compost
The chemical and physical characteristics of the produced biofertilizer are shown in   compositions showed significant (p < 0.05) changes in % moisture content on days 14, 28, and 42 of composting with all test groups, with A þ B showing the most significant (p < 0.05) changes. Also, there was a statistically significant (p < 0.05) downward trajectory in % organic carbon content as treatment days progressed, probably due to loss of carbon dioxide. Electrical conductivity (EC) decreased significantly (p < 0.05) through-out the fermentation process. Changes in the biofertilizer bacteria count were observed during the fermentation process. Biofertilizer A þ B showed the highest microbial count of 5.32 Â 10 9 CFU g À1 and 5.06 Â 10 9 g À1 on days 14 and 28, respectively. The lowest count of 4.53 Â 10 9 g À1 was observed on the last day of fermentation with biofertilizer A þ B. Biofertilizers A and B from day 0 to day 42 showed no statistical (p < 0.05) difference between them. With time, an increase in the microbial count was observed with all the biofertilizers, except for the A þ B biofertilizer, which showed a significant (p < 0.05) decrease only on day 42. Using two organisms could be the reason for this, as both organisms could complement each other in some way to achieve faster waste digestion.

Effect of biofertilizers on plant growth in pots
Phosphate and potassium biofertilizers used singly and combined improved test plants' growth parameters ( Figures  4-6). The greatest shoot heights at the end of the experiment (week 8) were obtained from treatment using biofertilizer formulation A þ B with values of 116 cm, 54.7 cm and 35.4 cm for Z. mays, S. lycopersicum and A. hypogea, respectively. Treatment A þ B also produced the highest leaf numbers of 13, 72 and 195; leaf widths of 8.6 cm, 3 cm, and 3.2 cm and leaf lengths of 83.6 cm, 6.4 cm, and 5.4 cm for the three plants, respectively. These are also reflected in the dry weight of the plants. Thus, all the treatments significantly (p < 0.05) increased the dry weights of Z. mays, S. lycopersicum and A. hypogea. Biofertilizer A þ B produced the highest increases in plant dry weights, equivalent to 2.32-, 2.26-and 1.66-folds increase on the control for S. lycopersicum, A. hypogea and Z. mays, respectively (Table 3). Principal component analysis of the growth impacts showed that PC1 is highest at 11.010, corresponding to 91.7% of the total variability (Table 4). This implies that the PC1 axis alone is sufficient to see 91.7% of the total variability of the data. Other PCs had eigenvalues (<1); hence, they will not be retained according to the Kaiser-Guttman criterion for retention (Kaiser 1960). On the PC1 axis, S. lycopersicum leaf number (SlLN) was the highest eigenvector. A. hypogea leaf number (AhLN), Z. mays leaf length (ZmLL), Z. mays leaf number (ZmLN) and A. hypogea leaf width (AhLW) were the highest eigenvectors under PC2, PC3, PC4, and PC5 respectively, thus, contributing to 3.8%, 1.9%, 1.2% and 0.6% variability in the data respectively. These make the study's five variables the most impacted by biofertilizer treatments.

Colonization and establishment of biofertilizer organisms in test crop rhizosphere
The use of biofertilizers product in pot growth of test crops led to the successful establishment of biofertilizers (P. aeruginosa and B. cereus) in the rhizosphere of all three plants. The highest recovery of biofertilizer microbial count, 2.89 Â 10 8 cfu/g (being the total count of the mixed population), was obtained from treatment A þ B for S. lycopersicum and Z. mays, while the least number 2.73 (10 8 cfu/g) was obtained from treatment A for A. hypogea (Figure 7). There was a statistically significant (p < 0.05) difference in the microbial count of the different treatment pots except for S. lycopersicum and Z. mays (A þ B). Growth of A hypogea, a legume, is strongly associated with very proficient nitrogen fixing PGPB. Although initial planting was done on sterile soil, these would likely have entered and associated with this plant's roots to impact other organisms' ability to establish in the rhizosphere. Different plants' physiological peculiarities can also influence biofertilizer microbes' ability to associate and establish in the rhizosphere.

Discussion
The use of microorganisms as biofertilizers for crop production is gaining traction globally (Rashid et al. 2016;Mitter 2021) and the market for it is expected to hit US$1.8b in 2025 following an annual growth rate of 14% from 2015 to 2020 (Daniel et al. 2022). These microorganisms are valuable for crop production because they increase plant growth and productivity by enhancing nutrient bioavailability (N, P, K, S, Zn) in a sustainable manner consistent with environmental protection and a green economy. They can do this because of their ability to mineralize and solubilize these nutrients from abundant but bound or otherwise unavailable organic or inorganic forms, a consequence of their natural involvement in biogeochemical cycles. They may also selectively and competitively protect plants from root pathogens and play roles in producing phytohormones that impact plant growth. Biofertilizers are produced and used in a variety of forms. They may be produced and used as pure culture in dry, liquid, or slurry form to colonize the rhizosphere. This approach is implemented where the crop is grown in soil bearing a non-available form of the target macro-nutrient. The use of rice husk and bones as sources of phosphorus and calcium in crop production has been reported (Fiameni et al. 2021). However, large-scale use of these refuse in crop production is constrained by the limited availability of the nutrients to plants necessitating their use with microorganisms (biofertilizers) that are able to mineralize and make the associated nutrients available to plants. Therefore, this is the basis for using this agricultural refuse for preparing biofertilizers for plant growth, achieving the triple purpose of providing nutrients, carrier for biofertilizers and reuse of waste.
In this study, out of 45 organisms recovered from the rhizospheres of Z. mays, S. lycopersicum and A. hypogea, seven isolates were phosphate solubilizing bacteria (PSB) while six isolates were potent potassium solubilizing bacteria (KSB). The solubilization indices for phosphate and potassium were high and compared favorably with data published for commercially produced biofertilizer organisms (Linu et al. 2019). The growth of these PGPB and the solubilization of phosphate and potassium were associated with a decline in the pH of the corresponding media from neutral values to acid (pH 4.3), comparable and similar to the report of Khanghahi et al. (2021). The decrease in medium pH following the cultivation of PGPB is associated with the production of different organic acids by the organisms, which aid in the solubilization of mineral phosphate and potassium solubilization (Zaidi et al. 2009;Lima et al. 2010). This may explain why the growth of potent solubilizers in this study was associated with a decline in the media's pH and digesting compost, while poor solubilizers failed to significantly reduce the pH of the compost (Zaidi et al. 2009). Acidic exudation products of the growth of PGPB have been shown to aid the release of phosphate and potassium from rocks such as mica (Zhang et al. 2013).
The six selected isolates that produced the highest phosphate and potassium solubilization were identified and genomic details were deposited with NCBI. As identified in this study, Citrobacter farmeri and Bacillus cereus have been demonstrated as potent macro nutrient solubilizers. They  have even been used successfully as commercial phosphate biofertilizers and biopesticides to achieve sustainable and enhanced rice production (Habib et al. 2015). Similarly, P. aeruginosa, also isolated in this study, has been demonstrated to promote plant growth by solubilizing potassium and producing indole acetic acid (IAA) in the rhizosphere of various crops (Linu et al. 2019). As with electrical conductivity, this study's % moisture content decreased as composting days continued. This could be due to evaporation (Gebeyehu and Kibret 2013), considering that this study, as is typical, was not carried out in moisture regulated environment. A decrease in moisture content can become necessary in this technology since it reduces product bulk and is expected to improve the survival or endurance of the PGPB in carrier compost. The electrical conductivity of biofertilizer group A þ B on day 42 (A þ B42), the final day of fermentation, had the lowest value of 280.00 ± 0.00 (uS/cm). This happened despite the significant increase in the different biofertilizer groups' percentage N, K, and P content during fermentation. This conductivity profile is consistent with a decrease in the moisture content of the compost and compares well with results obtained by Hamouda (2016). It accounts in part for the drop in pH that occurs during a period of active microbial growth in the fermentation process, as has been alluded to by (Kartini and Dhokhikah 2018) in a comparable reaction environment. The pH of compost is used as an indicator of compost maturation and stabilization process (Gebeyehu and Kibret 2013), often rising to settle between 7.0 and 8.0 at the end of the process, as also obtained in this study. The rise in pH to neutral and alkaline at the end of the reaction is consistent with compost behavior and particularly suited for the survival of PGPB in the product, long enough to establish in the rhizosphere and during storage of the product prior to application.
The association between biofertilizer treatments and development characteristics of three different crops S. lycopersicum, Z. mays, and A. hypogea, were investigated using principal component analysis (PCA) ( Table 4) of plant development data for shoot height, leaf number, leaf width, and leaf length. PC1 and PC2 explained 91.7% and 3.8% of the total variance. On the PC1 axis, the highest eigenvector was S. lycopersicum leaf number (SlLN), implying that the different biofertilizer treatments had the most significant impact on S. lycopersicum leaf number (SlLN). Under PC2, PC3, PC4, and PC5, the highest eigenvectors were A. hypogea leaf number (AhLN), Z. mays leaf length (ZmLL), Z. mays leaf number (ZmLN), and A. hypogea leaf width (AhLW), contributing 3.8%, 1.9%, 1.2% and 0.6% variability in the data respectively. Biofertilizer treatments significantly impacted these key plant development variables and are expected to translate to increased crop productivity. Biofertilizers, particularly non-nitrogen fixing bacteria, are known to differently and often selectively impact crops, with plant specificity being fairly common as reported with Z. mays (Schmidt and Gaudin 2018); A. hypogea and Z. mays (Anzuay et al. 2017); and S. lycopersicum (Coulibaly et al. 2021).
Results of one-way analysis of variance (ANOVA) of the effect of biofertilizers; A, B and A þ B, and C treatments on plants' dry weights (   resulted in the highest increase in dry weight of Z. mays. Hindersah et al. (2021) found that using Bacillus spp. biofertilizer to grow nutmeg seedlings in pot culture improved growth. Similarly, Etesami et al. (2017) found increased tissue dry weight and nutrient uptake in black pepper and increased grain yield, shoot mass, and nodule mass in chickpea following biofertilizer application. The A þ B treatment resulted in the most significant increase in S. lycopersicum and A. hypogea dry weight. The work reported here is interesting for the consistency of impact of the biofertilizers used in this study on crops from different groupings, including legumes, grass and vegetables. At the end of the experiment, the inoculants (P. aeruginosa and B. cereus) demonstrated effective rhizosphere colonization. Treatment A þ B (S. lycopercicum) had the highest Bacillus cereus colonization with a microbial count of 2.89 Â 10 8 cfu/g, whereas treatment A (A. hypogea) had the lowest microbial count of log 2.73 Â 10 8 cfu/g. In all three test crops, the plants inoculated with combined phosphate and potassium biofertilizer (A þ B) showed a more significant growth impact than the single treatment or the control. The two organisms used in the treatment A þ B complemented each other in improving plant growth and suggest a possibility for broad-acting biofertilizers when consideration is given to the carrier material. However, it is not clear if their actions were synergistic. The actions of the two organisms could have been additive, although the possibility of synergy could not be excluded. The result of this study is interesting because it was implemented in a screen house, as opposed to the climate-controlled greenhouse of many studies. It is encouraging, therefore, that comparable performance can be expected to obtain in related farm situations. A significant challenge with research on PGPB has been the inability to replicate greenhouse performance in the field (Mitter et al. 2021), particularly as it impacts the establishment and persistence of the candidate organisms in the rhizosphere. This has been demonstrated to be overcome in this study.

Conclusion
The use of PGPB singly and in particular combinations and consortia is gaining traction for sustainable crop production, particularly in challenged soil systems. This development could be of strategic food security interest in tropical developing countries with limited chemical fertilizer access and dependent on rain-fed crop production systems. In this study, the application of locally isolated PSB, Bacillus cereus (OM328186) and KSB, Pseudomonas aeruginosa (OM328188) biofertilizers has shown the potential to dramatically cause improvement in the growth and productivity of key tropical food security crops, including Z. mays, S. lycopersicum, and A. hypogea whose yields currently lag well behind the productivity of countries with developed agricultural systems. These isolates are used in combination to improve the growth of three unrelated crops; legume, grass and horticultural vegetable crop. The result of this study is quite interesting and could be leveraged to impact sustainable crop production in the soil types employed in this study. The result of this study is also noteworthy for its utility in valorizing cattle bone and rice husk (two wastes currently considerable nuisance) in agricultural production.