1 Introduction

Bacteria colonization and biofilm development on a material begin with an initial adhesion. Biofilms may be harmful to human health and industrial operations, producing infection, pathogen contamination, and slime production, for example, while also being useful in environmental technologies and bioprocesses. Wild-type bacteria have evolved numerous cooperative skills, such as the capacity to swarm or form surface-attached colonies known as biofilms, as a result of their evolution in changing settings and competition for limited resources. These  'city of microbes' are created in response to a variety of stimuli and have been demonstrated to give an advantage in a variety of scenarios, ranging from nutrition retention to predator protection via greater antibiotic resistance (Moradali and Rehm 2019). As the biofilm matures, its inhabitants begin to generate an adhesive matrix, which allows cells to adhere to one another and form a layered biofilm. The matrix, also known as extra polymeric material (EPS), is mostly made up of exopolysaccharide, protein, and DNA (Hamon and Lazazzera 2001; Abdullah et al. 2021).

The capacity of creating a biofilm relies on the presence of bacteria that can release extracellular polymeric substances (EPSs), which  'glue' cells together and onto the surface, inside the community (Dervaux et al. 2014). Adhesion mechanisms must be explored in order to regulate and use bacterial adhesion and biofilms (Ajeng et al. 2021c, 2021a). In the development of a biofertilizer, the selection of bacterial species is an important component towards a successful intended applications in the soil. However, due to the heterogeneity of soil microbes and resident pathogens, the intended application of biofertilizer without any carrier is often unsuccessful due to the grazing by the pathogen and competition with the resident soil microbes.  That is why the recent emerging biofilm immobilization on a carrier method is becoming popular for these reasons.

Biofilmed Biofertilizer (BBF) is an emerging technique in biofertilizer development which is based on the biofilm formation of beneficial plants probiotics on certain carrier which allows for a safe delivery into the soil, to avoid grazing by soil pathogen and competition with resident soil microbiota (Dervaux et al. 2014; Otaiku  et al. 2022). B. subtilis has been used to research Gram-positive bacterial physiology as a model organism. B. subtilis produces adherent biofilms on inert surfaces under the influence of a number of transcription factors, according to early studies (Hamon and Lazazzera 2001; Stanley et al. 2003). There is a clear recognition that biosurfactant synthesis not only influences biofilm architecture but also influences bacteria adhesion to surfaces (Davey et al. 2003). The effectiveness of the biofertilizer often depends on several factors including types and abundance of carbon and nitrogen sources, temperature, pH, aeration, and incubation period (Barin et al. 2022). Therefore, different formulations are optimized following these factors, in order to have the most efficient biofertilizers.

Biochar is an established organic soil amendment produced via the pyrolysis of biomass under limited oxygen (Lehmann and Joseph 2015; Ajeng et al. 2020). The growing evidence of biochar-immobilized microbes in various biotechnological sectors such as the bioremediation provides better insights into the interaction between the materials and the microorganisms (Li et al. 2022). However, the persistence of the biofilm generated on biochar and the subsequent evolution of the immobilized microorganisms received little attention particularly in the biofertilizer development. When developing a bioformulation with any type of carrier material, several factors governing bacterial growth such as pH, temperature, culture medium, and time should be considered to achieve an optimised microbial growth with viable cell count that is within the minimum range of 106—107 CFU per gramme of the material (Vanek et al. 2016; Abdulrasheed et al. 2020). Many variables impact the physical foundation of bacterial biofilm development on surfaces such as biochar, including electrostatics, metal ion concentration, carrier zeta potential, and bacterial motility. According to Hong et al. (2012), hydrophobicity exhibited no discernible relationship with other adhesion characteristics, however the surface electrical property dominated the affinity between Bacillus subtilis and six soil minerals. On the other hand, Bejarano et al. (2017) showed that the zeta potential had only a little impact on the effectiveness of Paraburkholderia phytofirmans PsJN's adsorption on talc and a small number of other carriers. According to Zhang et al. (2021), bacteria from activated sludge are more likely to form biofilms on cationic polyacrylamide-derived hydrophilic group-modified material. According to Bejarano et al. (2017), bacteria can build up more readily on hydrophobic carriers than on hydrophilic mineral surfaces, which is the case for most biochars. Furthermore, a new study shows that single bacterial species may be immobilized on biochar for biotechnological purposes rather than a community of bacteria, which is required in the production of an efficient biofertilizer(Cheng et al. 2020; Ajeng 2021). Thus, the aim of this study is to optimize the operating conditions for the immobilization of Bacillus consortium biofilm formation on palm kernel shell biochar. We hypothesized that addition of co-carbon source such as starch during the optimization process would positively affect the immobilization and encourage biofilm formation on palm kernel shell biochar.

2 Materials and methods

2.1 Chemicals and reagents

Phosphate buffered saline (PBS)(Sigma Aldrich), Brain heart infusion (BHI)(Sigma Aldrich), Sago Starch (Food Grade), sodium hydroxide (NaOH) tablets (Sigma Aldrich), 5% hydrochloric acid (HCl) (Ajeng et al. 2021c).

2.2 Biochar preparation and Bacillus consortium preparation

Oil palm kernel shell biochar (OPKSB) was obtained from Malaysia Palm Oil Berhad (MPOB) which was produced via slow pyrolysis process at 400 °C. The characterization and properties of OPKSB were described previously (Ajeng et al. 2021c). OPKSB biochar was rinsed with water to remove water soluble fractions which could potentially possess toxicity towards microbes. The OPKSB was then dried in an oven at 100 °C to remove remaining moisture prior to grinding into coarse particles using pestle and mortar (Vejan et al. 2022). The ground OPKSB was sieved to pass a 2 mm sieve.

Bacillus Strain Salmah Ismail (SI) 139SI was obtained from the Universiti Malaya Molecular Bacteriology and Toxicology Laboratory (UMBTL) and was first isolated from soil taken from a private farm in Selangor, Malaysia (2.99917°N 101.70778°E) (Ismail and Dadrasnia 2015). Meanwhile, B. amyloliquefaciens NBRC15535 and B. cereus ATCC14579 were isolated from a biofertilizer for oil palms (Isolation and identification data  were shown). These strains were routinely cultivated on BHI blood agar and kept at -80 °C in a glycerol solution (25% w/v) and at ambient temperature on BHI-slant agar. In vitro assays were used to determine the compatibility of bacterial species, according to Sundaramoorthy et al. (2012) (Sundaramoorthy et al. 2012). Cell suspensions were prepared and spread on BHI plates in all possible combinations  of the three species. In relation to each other, each pair of species was spread out in perpendicular lines. Plates were incubated for 72 h at 28 °C before being examined for inhibition zones. Compatible strains were able to grow over each other, and inhibition zones present between incompatible combinations. The experiment was repeated twice for each culture medium, with four replicates per treatment. All three Bacillus species were compatible with each other (Fig. 1). A standard consortium suspension was then prepared by mixing equal volume from each Bacillus grown in BHI medium (1:1:1) into sterile PBS (Ajeng et al. 2021c).

Fig. 1
figure 1

From left: B. salmalaya against B. cereus, B. salmalaya against B. amyloliquefaciens and B. cereus against B. amyloliquefaciens, respectively

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2.3 Optimization of operating conditions on Bacillus consortium survivability

The Wijesinghe et al. 2015 (Wijesinghe et al. 2019) method was used to conduct the biofilm growth assay with slight modification. To initiate the first cell adhesion on OPKS biochar, 1000 µL of the standard cell suspension was pipetted into sterilized conical flask containing 0.25 g OPKS biochar and statically incubated for 90 min at 37 °C. The flask was then washed once with 2000 µL of sterile PBS to remove non-adherent cells before being replenished with 1000 µL of sterile BHI broth.

The optimization of Bacillus consortium biofilm adhesion on OPKS biochar was conducted using Response Surface Methodology (RSM) technique. A central composite design (CCD) was applied to find the interaction of each parameter with a response (Design-Expert 13, Stat-ease, Minneapolis, USA). The Bacillus population (CFU/ml) was chosen as the response for this study (Eq. (1)) due to the intended bioformulation application as soil organic amendment where cell viability matters the most in designing an effective carrier (Ajeng et al. 2020; Mitter et al. 2021).

$$\mathrm{Colony Forming Unit }(\mathrm{CFU}/\mathrm{mL})= \frac{TC \times df }{V}$$
(1)

where TC is the total colonies, df is the dilution factor and V is the volume plated in mL.

The Bacillus population was determined using the mathematical model designated by Eq. (2).

$$y= \beta 0 + \sum_{i=1}^{n}\beta i\chi i +( \sum_{i=1}^{n}\beta ii\chi i {)}^{2} +\sum_{i=1}^{n}\sum_{i\ne j}^{n}\beta ij\chi ij$$
(2)

where \(y\) is the response of interest (CFU/mL), \(\chi i\) and \(\chi j\) are the parameters (Temperature (°C), agitation speed (rpm), pH, and starch (% w/w), and \(\beta\) is the regression coefficient value of the model. The pH of the medium was adjusted using NaOH or HCl. The results were statistically analyzed using analysis of variance (ANOVA) with p-value < 0.05 to determine optimized Bacillus population. The ranges and levels of independent variables (parameters) are list in Table 1. The conditions of the experiments are shown in Table 2.

Table 1 Ranges and levels of independent variables used for the experimental design of Bacillus consortium biofilm adhesion on OPKS biochar
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Table 2 Summary of population for Bacillus consortium from biofilm adhesion on OPKS biochar
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2.4 Bacillus consortium adhesion kinetics modelling

The adsorption kinetics was investigated with initial concentrations of 3.0 \(\times\) 108 CFU mL−1 during incubation periods of 0 to 140 min at optimal conditions defined by the RSM analysis. The 100 \(\mathrm{\mu L}\) of the test solutions were centrifuged (3000 rpm) for 5 min (Du et al. 2016) and the supernatant was the unadhered bacteria. The CFU/mL was calculated following Eq. (1) and the attached bacteria was calculated by subtracting the initial concentration with the obtained values. In the current study, three linear kinetic models, namely pseudo-first order, pseudo-second order, and intraparticle diffusion model, were used to investigate the adsorption dynamics of Bacillus spp. onto PKS biochar, where the models allow estimation of the number of bacteria absorbed at the time of processing. The kinetic models also allow for the estimate of sorption rates, which frequently leads to rate expressions that are indicative of putative adhesion processes (Robati 2013). The linearized pseudo-first order (PFO) is expressed as follows:

$$\large \tiny {log }({Q}_{e}-{Q}_{t})=\mathrm{log K}Qe-\mathrm{K1t}$$
(3)

where Qe (CFU g-1) and Qt (CFU g-1) represent the adsorbate quantity adsorbed at equilibrium and at any t (min), respectively; K1 is the rate constant of adsorption (1 min-1). The value of  K1 and Qe were determined from the slope and the intercept of the log (Qe–Qt) against t graph. By drawing the diagrams of log (Qe−Qt) and t/qt against time, the constant parameters of pseudo-first and pseudo-second order kinetic models may be determined and correlation coefficient R2 may also be calculated for laboratory conditions.

The linearized pseudo-second order (PSO) is expressed as follows:

$${\frac{t}{{Q}_{t}}}= {\frac{1}{K2{Q}_{e}^{2}}}+{\frac{t}{{Q}_{e}}}$$
(4)

where K2 is the rate constant of the adsorption (CFU/g min). The value of  Qe and  K2 were determined from the slope and the intercept of the t/Qt against t graph. To compare the efficiency of the kinetic models in describing the adhesion properties, non-linear models of each of the kinetic model were utilized. The non-linear PFO is expressed as follows:

$${Q}_{t}= {Q}_{e} (1-{e}^{-kt})$$
(5)

where Qe and Qt are the adsorbate uptake rates per mass of adsorbent at equilibrium and at any time t (min), respectively, and k (min−1) is  the rate constant of PFO equation.

And non-linear PSO is expressed as follows:

$${Q}_{t}= \frac{{K}_{2}{Q}_{e}^{2}t}{1+ {K}_{2}{Q}_{e}t}$$
(6)

where Qe (CFU g-1) and Qt (CFU g-1) represent the adsorbate quantity adsorbed at equilibrium and at any t (min), respectively, and K2 (g/CFU min) represents the PSO equation constant rate.

2.5 Bacillus consortium Sago Starch-biochar Adhered Characterization and Storage Potential

The optimized formulation was replicated and left to dry in an aseptic container wrapped in aluminum foil. Equal volume of the formulation was stored in different temperatures (Sun et al. 2016) (-24 °C, 32 °C, and 39 °C) for 4 months and sterilized biochar without inoculants acted as the control. An in vitro soil incubation was also performed following the methods outlined by Husen (2003) with slight modifications. Briefly, three sterilized glass jars containing oxisol  were inoculated, mixed with 10% (w/w) of the optimized formulation and kept slightly aired. The water holding capacity of soil was capped at 70% by using sterilized distilled water (Sarkar et al. 2005). The jar was kept at room temperature for 4 months. To obtain bacterial isolations, the protocol described by Mandakovic et al. (Mandakovic et al. 2018), previously used to isolate bacteria from soil samples in the Lejía Lake, was followed with slight modifications. 1 g of the formulation (storage potential) and 1 g of soil (in vitro incubation)  were diluted in 900 \(\mathrm{\mu L}\) of sterilized distilled water and vortexed. 100 \(\mathrm{\mu L}\) of the suspension  were diluted to a dilution factor (df) of df = 3 or df = 4, and 30 \(\mathrm{\mu L}\) of the final dilution was pipetted on three BHI plates and incubated overnight. The total viable bacteria count was performed using Eq. 1. The results  were presented in CFU/ml and visual plates images. Fourier transformed infrared (FTIR) for OPKS biochar  served as the control and OPKS biochar adhered Bacillus consortium were conducted. Prior to storage, the formulation was freeze dried and sent for Field Emission Scanning Electron Microscope (FESEM) analysis to visualize the adhesion of cells onto the OPKS biochar.

2.6 Statistical analysis

Experimental data  were statistically analyzed using SPSS statistical software (version 19.0, IBM Corp., Armonk, USA). The results for optimization were statistically analyzed using analysis of variance (ANOVA) with p-value < 0.05 to determine optimized Bacillus population. The multiple regression analysis of the model and construction of response surface graphs were performed by using Design-Expert, version 7.1.6 (STATEASE Inc., Minneapolis, MN, USA). The quality of regression equation was determined by the coefficient of determination R2 and its significance was judged by an F-test. The fitted 2nd order polynomial equation was explained in the form of 3D plot graphs to show the relationship between the response and experimental variables. The point optimization method was used to optimize the maximum response of each variable. Moreover, in order to prevent systematic errors, the run order of experiments was randomized. Differences in the bacteria survivability were evaluated by one-way ANOVA (p < 0.05).

3 Results and discussion

3.1 Optimization of Bacillus consortium biofilm adhesion on OPKS biochar using RSM analysis

Bacillus biofilm adhesion on the biochar was performed under different operating conditions (temperature, pH, agitation speed and starch concentration). The optimization of these parameters was conducted to examine the conditions which promoted high bacterial growth and survivability (high CFU/mL) (Fig. 2). Therefore, the basic Bacillus population (CFU/mL) was chosen as a response for CCD analysis. The Bacillus population is presented in Table 2. The model for the relationship of parameters X1, X2, X3, and X4 on Bacillus consortium survival (Y) is shown in Eq. (7). The statistical significance and the goodness of fit for bacterial survival related to the parameters were proven by the R2 and p-values. It was found that the R2 (0.998) of the model was close to R2 adj (0.996). The p-value of lack of fit was higher than 0.05. These statistical values confirmed that the model had adequate precision and was reliable for modeling the Bacillus consortium survival from biofilm adhered on the OPKS biochar.

$$\mathrm{y}=2.31\mathrm{E}8-4.79\mathrm{E}6 {x}_{1}+1.44\mathrm{E}6{x}_{2}+ 5.00\mathrm{E}5{x}_{3 }+ 3.35\mathrm{E}5{x}_{4 }+2.917\mathrm{E}5{x}_{1}{x}_{2}- 5.42\mathrm{E}5{x}_{1}{x}_{3}+4.17\mathrm{E}5{x}_{1}{x}_{4} +7.50\mathrm{E}5{x}_{2}{x}_{3}+5.83\mathrm{E}5{x}_{2}{x}_{4} +4.17\mathrm{E}5{x}_{3}{x}_{4}-1.73\mathrm{E}5{x}_{1}^{ 2}+8.19\mathrm{E}5{x}_{2}^{ 2}-2.10\mathrm{E}8{x}_{3}^{ 2} -4.97\mathrm{E}6{x}_{4}^{ 2}$$
(7)

where  y is Bacillus adhesion rate (mg L−1d−1) and X1, X2, X3, and X4 are temperature (°C), agitation speed (rpm), pH, and co-carbon source concentration (%w/w), respectively. This indicates that the alternated model was effective at estimating the Bacillus adhesion. The equation in terms of coded factors may be used to predict response for different amounts of each parameter. The high values of the components are recorded as + 1 by default, while the low levels are coded as -1. By comparing the factor coefficients, the coded equation may be used to determine the relative importance of the components. The F-value of 531.45 for the model indicates that it is significant. Model terms are significant if their p-values are less than 0.05. Significant model terms in this scenario are X1, X22, and X32. The model terms are not significant if their values exceed 0.10. Model reduction may enhance this model if there are a lot of unimportant model terms (except those necessary to maintain hierarchy). The F-value of 0.96 for the  lack of fit indicates that it is not significant when compared to the pure error. The model has a significant   lack of fit F-value due to noise, which has a 55.58% probability of occurring.

Fig. 2
figure 2

The interaction of various operating factors on the immobilization of Bacillus consortium biofilm on palm kernel shell biochar

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The CCD results of Bacillus population released from the maturation biofilm were analyzed by the ANOVA test. At the 95% confidence range, only the temperature and starch % (w/w) had statistically significant effects on Bacillus survival rate with other parameters showing no clear influence. Low to intermediate operating temperature significantly increased the Bacillus population. This indicates that the biochar-adhered bacterial biofilm is effective in releasing bacterial cells after biofilm maturation within the medium range of temperature tested whereby the highest temperature of 38 °C is detrimental to the bacterial biofilm adhesion, which leads to decreased bacterial survival on biochar ecosystem.

The findings were in agreement with previously published studies (Hoštacká et al. 2010; Rao 2010; Pavlovsky et al. 2015) which suggested that the bacterial biofilm formation is optimum at temperature ranging from 30 to 33 °C. In addition to EPS generation, a second change  occurred during biofilm formation, which was flagella synthesis downregulation.  de Kievit (2011) suggested that there is an inverse link between EPS production and fla gella through a transcriptomic analyses, which demonstrates that genes involved in the creation of the biofilm matrix and flagella are inversely expressed. The findings from these studies will add to the current knowledge repositories on the effects of temperature on bacterial biofilm formation on material surfaces, where in the medical and food industries, preventing the formation of pathogenic bacterial biofilm is of major important. Meanwhile, the development of bioformulation containing beneficial inoculants intended for agricultural purposes requires a substantial amount of the cells to be safely delivered into the soil to allow a successful interaction with plant roots (Vanek et al. 2016; Ajeng et al. 2020; Egamberdieva et al. 2020; Saleh et al. 2020). It is interesting to note that addition of sago starch as co-carbon source for the Bacillus  has a positive effect on the survival of the consortium on OPKS biochar. It is believed that the addition of starch as co-carbon source in the growing medium of the bacteria  increases the survival of the strain due to the induced production of biosurfactant (Stancu 2020). Biosurfactant is an emerging useful biotechnological compound secreted by Bacillus species that aids in facilitating the transport and translocation of insoluble hydrophobic substrates (e.g., polyaromatic hydrocarbons, and oils) across the cell membranes (Dadrasnia and Ismail 2015; Stancu 2020). Furthermore, these biocompounds can withstand a broad variety of pH and temperature conditions, and they can influence interfaces to some extent (Diniz Rufino et al. 2014). The potentials of B. salmalaya 139SI in biosurfactant production have been evaluated in several studies (Dadrasnia and Ismail 2015; Ismail and Dadrasnia 2015; Abu Tawila et al. 2018). However the growth and production of biosurfactant from this species in starch addedstarch-added medium have not been reported. From our findings, the addition of sago starch by 20% (w/w) slightly increased the survival of the consortium in low pH and high temperature. Meanwhile, 10% (w/w) of starch addition is adequate to improve the growth of Bacillus consortium on OPKS biochar (108  CFU ml-1).

Other parameters, on the other hand, had a less influence on Bacillus consortium population (agitation and pH). The parameters that were chosen for optimization study were based on real-world operational and environmental circumstances. However the ranges may not be critical, resulting in a negligible effect on Bacillus population from the adhered biofilm. According to Table 2, an increase in Bacillus population was observed in neutral pH (7.5), with less growth noticed in pH 3 and pH 12. Enhanced bacterial growth in neutral pH ranges could be due to the higher alginate exopolysaccharides production (Boyd and Chakrabarty 1994; Moradali and Rehm 2019). These natural polymers are important polymeric substances contributing to the formation and development of biofilm matrixes of numerous bacteria enhancing their persistence under various environmental stresses (Ajeng et al. 2021b; Ismail et al. 2022).

3.2 Kinetics properties of Bacillus Consortium on OPKS Biochar and its adhesion characterization

From the FESEM analysis shown in Fig. 3, a network of rich Bacillus biofilm and formation of Bacillus colonies on OPKS biochar can be observed. The biofilm is seen to encapsulate the biochar surfaces (Fig. 3c) including its pores. The bioencapsulation of carrier material is preferable in the development of an inoculant carrier for soil application over alternative solid and liquid formulations (Schoebitz et al. 2013). The problems pertaining to the carrier material such as the ineffectiveness to deliver substantial amount of biofertilizer into the soil can be addressed by the immobilization microbe biofilm-based formulations (Vassilev et al. 2020). Therefore, this biofilm-biochar technology is deemed to revolutionize not only the wastewater treatment and bioremediation but also the biofertilizer technology over the conventional formulation types (Dalahmeh et al. 2019; Jayakumar et al. 2021; Xing et al. 2021; Ajeng et al. 2022).

Fig. 3
figure 3

FESEM of a OPKS biochar, b–d Bacillus consortium biofilm adhesion on OPKS biochar and e The FTIR analysis of OPKS biochar and OPKS biochar adhered Bacillus consortium

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In a study conducted by Ajeng et al. (2021c), the author suggested that oxygen-containing functional groups play an important role in polymer adherence to surfaces. EPS are polymers produced by a variety of bacteria, including the Bacillus species employed in this work. The formation of EPS was demonstrated by the FTIR measurements in Fig. 3e. The -1,4-glycosidic bond, amide II, and uronic acids (1600 – 1200 cm−1) were identified as functional groups corresponding to Bacillus EPS production potentials in the investigated spectra. These peaks were not seen in the OPKS biochar-only as the control sample. The maxima of the amide I band, generally found at around 1650–1660 cm−1, shows the prevalence of helices among the secondary structural components of bacterial cellular proteins under normal growth circumstances (Kamnev 2008). Meanwhile, the functional groups of polysaccharides and nucleic acids are exhibited by the FTIR spectra in the 900–1,300 cm−1 range.

Non-linear and linear kinetic models were used to determine the adhesion kinetics of Bacillus consortium on the OPKS biochar. The bacteria adhered quickly in the first 60 min, and the greatest adhesion was attained after 120 min when the bacterium concentration was 2.0 × 108 CFU g-1 (Fig. 4). In comparison, the linear pseudo-second order kinetic model describes the adhesion kinetics of the Bacillus consortium well on OPKS biochar. For the adhesion system with contact durations of 140 min (Fig. 4), the correlation coefficients for the linear plots of t/qt against time from the pseudo-second order rate law are more than 0.998. This implies that the sorption system is not a first order reaction, and that the pseudo-second order model, based on the assumption that the rate-limiting step may be chemical sorption or chemisorption. The adsorption may involve valency forces via electron sharing or exchange between sorbent and sorbate, providing the best data correlation (Ho and McKay 1999) where the rate-limiting step is postulated to be the biofilm diffusion.

Fig. 4
figure 4

Experimental data and the fitted non-linear (a) and linear (b, c) PFO and PSO models, respectively

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3.3 Storage potential and in vitro soil incubation of optimized formulation

The limited shelf life of inoculants continues to stymie biofertilizer technologies (Aloo et al. 2022) in which the selection of a better inoculant carrier that can deliver a substantial amount of probiotics or beneficial microbe to the soil is of importance (Ajeng et al. 2020). The Bacillus population from the storage potential at different temperatures and in vitro soil incubation  are shown in Figs. 5 and 6, respectively and Fig. 7 shows the BHI plate conditions after incubations.

Fig. 5
figure 5

The storage potential of the optimized formulation in different temperatures

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Fig. 6
figure 6

The in vitro soil incubation of the optimized formulation

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Fig. 7
figure 7

The plate images from storage potential a control, b 32 °C, c 39 °C d -24 °C and in vitro soil incubation e R1, f R2 and g R3 of the optimized formulation after 3 months. Arrows indicating the Bacillus species

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It was evident from the storage potential results that the optimized formulation consisting of OPKS biochar loaded Bacillus was able to be stored for at least 3 months with substantial amount of live Bacillus (107  CFU g-1) which was above the minimum range of live bacteria within a biofertilizer (106  CFU mL-1) (Vanek et al. 2016). After 4 months, the formulation maintained in the freezer exhibited the lowest activity of Bacillus compared to the formulation stored at ambient temperature (32 °C) and at 39 °C. This could be due to the inactivation of cells caused by the low moisture content (MC) in formulation stored in the freezer (Aloo et al. 2022). The MC of carrier materials has a significant impact on inoculant survival and longevity. This is most likely due to the inoculants' ability to remain dormant, resistant to environmental stressors, and unaffected by contamination in carriers with low MC. Dry formulations with low MC may often extend microbial viability for longer periods of time and at higher temperatures, resulting in lower marketing and maintenance costs because refrigeration is not necessary. Meanwhile, the bacterial activity from the cold storage can be revived before inoculating the formulation as a soil amendment, however a repeated freeze–thaw process of this formulation could be detrimental to the survival of the Bacillus as reported by Walker  et al. (2006).

To further improve the storage potential, several manipulations to the bacterial strains can be made such as the genetic engineering of strain by introducing the cspB, which is the Bacillus cold shock gene responsible for the production of antifreeze proteins which protects the strain from the detrimental impact of ice crystallization in extreme cold storage temperature (Willimsky et al. 1992). Since the cspB gene and other antifreeze genes can be found in Bacillus species, the possibility of the horizontal gene transfer (HGT) to the Bacillus strains used in this study is higher and achievable although homogenization of HGT may occur in the field. Although high temperature can compromise the growth of bacteria in biofertilizer, OPKS biochar inoculated with probiotics biofilm can revolutionize the biofertilizer technology by providing sufficient surface area for the biofilm adhesion and protection against extreme environmental stressors. From the finding, formulations that have remained viable in room storage can be simply incorporated into current agricultural distribution systems that do not include refrigeration. The findings of our storage potential of beneficial biofilm loaded on biochar revealed that OPKS biochar is a promising choice as an inoculant carrier.

Another key criterion used to measure the performance of the formulation as a soil amendment is the in vitro soil incubation of the OPKS biochar loaded Bacillus in view of the bacterial survivability in a controlled soil incubation before inoculating them into the field. From the aerobic soil in vitro incubation, it can be noted that all  the three replicates exhibited a promising  shelf life in the soil for 4 months with a range of bacterial survival 107  CFU g-1 of soil. In comparison to the formulation, bacterial survival from the liquid formulation (control)  dropped at the second month of incubation, proving the efficiency of OPKS biochar as an inoculant carrier, providing surface adherence for these strains and protecting them against soil pathogen predation that may present even in an autoclaved soil medium (Baker et al. 2020). Thus, the optimized formulation may be suitable to be applied  in soil where there is a rapid accumulation of soil pathogens (Nijjer et al. 2007; Radian et al. 2022) or soil pollutants where OPKS biochar may safely offer a significant amount of probiotics to enrich the soil ecology and boost plant growth. This effect has been reported in the article of   Meng et al. (2019) where the author concluded that the addition of biochar improves the survival of the inoculant including the overall soil microbiome and wheat  plant performance under herbicide fomesafen stress. In another study by Naveed et al. (2020), the twigs biochar inoculated Burkholderia phytofirmans PsJN reduced the uptake and translocation of the heavy metals in the mung bean (Vigna radiata L.) plant. Future research relevant to the application of optimal formulation of OPKS biochar loaded Bacillus biofilm should include in vitro toxicity assessments of various heavy metals or herbicides, which will contribute to the knowledge repository from this work. Furthermore, according to Vanek et al. (2016) and Ajeng et al. (2020), the physicochemical properties of biochar are among the major factors affecting the potentials of biochar as carriers. The authors stated that the higher pore volume and surface area with functional groups that support the adhesion of microbes are of importance. The high porosity and large surface area of biochar offer a habitat for the development of immobilized microorganisms and protect them from soil faunal predators such as mites, protozoans, nematodes, and Collembola (Głodowska et al. 2017). Nano-pore carriers with a pore diameter of less than 25 nm may not be suitable for bacterial attachment due to the electrostatic force exerted on bacterial cells, as previously demonstrated on an anodic alumina surface, and biochar engineering may be required to ensure the porosity is suitable for the attachment of microbes (Feng et al. 2014). In the case of biochar surface properties, as additional oxygen functional groups are produced when the immobilized bacteria oxidize the biochar, stabilizing the soil's organic matter, adding more oxygen functional groups to the surface of the biochar may be advantageous for its surface qualities (Sun et al. 2016). The fused carbon rings in biochar support the microbial electron shuttle, redox reaction, microbial metabolism, and nutrient cycling. After biochar is pyrolyzed, these carbons, which also include other functional groups like oxygen and hydrogen, bond to the nutrients and minerals (Batista et al. 2018; Oh et al. 2018). Therefore, investigating the impacts of biochar features such as feedstock type, ash content, carbon content, pore and micropore volume, and surface functional group variation on Bacillus consortium adhesion to biochar may be a significant topic for future research.

4 Conclusion

This study presents the potential of Bacillus biofilm adhesion on OPKS biochar in view of the development of effective biofertilizer for soil amendment. The optimization of strain adhesion on the carrier material produced a favorable environment for consortium growth at 33 °C, 135 rpm agitation speed, neutral pH of 7.5, and 10% (w/w) sago starch as the co-carbon source in which may have assisted in the production of EPS from the Bacillus species used in this study. The optimized operating conditions from this study may be replicated for future work involving its application as biofilm-biochar biofertilizer to enhance crops growth. Bacillus biofilm-OPKS biochar may survive up to 4 months in storage and in vitro soil incubation, indicating that there is an adequate number of probiotics released by this formulation.