DOI: 10.3303/CET24111123 Paper Received: 27 March 2024; Revised: 29 May 2024; Accepted: 27 June 2024 Please cite this article as: Maumela P., Serepa-Dlamini M.H., 2024, Compost Biostimulation for Improved Soil Bioremediation Using Endophytic Bacillus Sp., Chemical Engineering Transactions, 111, 733-738 DOI:10.3303/CET24111123 CHEMICAL ENGINEERING TRANSACTIONS VOL. 111, 2024 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Valerio Cozzani, Bruno Fabiano, Genserik Reniers Copyright © 2024, AIDIC Servizi S.r.l. ISBN 979-12-81206-11-3; ISSN 2283-9216 Compost Biostimulation for Improved Soil Bioremediation using Endophytic Bacillus Sp. Pfariso Maumela*, Mahloro H. Serepa-Dlamini University of Johannesburg, South Africa pmaumela@uj.ac.za The use of bacterial endophytes in soil bioremediation is limited by nutrient availability in the soil. In this study, garden compost was used as a biostimulant during the bioremediation of lead contaminated soil with a bacterial endophyte. The experimental data showed that the cell wall of the Bacillus sp. strain MHSD_36 resulted in 36% lead biosorption. The addition of glucose and peptone in the growth media, enhanced biomass growth and lead biosorption, achieving a maximum of 76% lead removal. The use of compost as a cheaper carbon and nitrogen source improved the biosorption capacity of strain MHSD_36 during the bioremediation of lead contaminated soil. Compost biostimulation resulted in a residual lead of 250 mg/Kg, from an initial concentration of 300 mg/Kg, compared to 282 mg/Kg without biostimulation. The endophytic bacteria Bacillus sp. strain MHSD_36 is a potential lead biosorbent. Moreover, compost is an organic biostimulator with potential application in soil decontamination. 1. Introduction The fast growth nature of bacteria and ability to adapt their growth strategies in harsh environments has accelerated their interest and use in bioremediation (Pande et al., 2020). Bacteria possess an array of mechanisms for the detoxification of hydrocarbons and heavy metals because they carry a diverse set of catabolic genes and enzymes (Pal et al., 2022). Bacteria synthesize and secrete surface active compounds such as siderophores and biosurfactants and use efflux pumps to remove toxic compounds from the cytoplasm to the periplasm and environment (Presentato et al., 2020). Bacteria can modify their cell membrane to maintain biological function and survive in contaminated environments (Dell’Anno et al., 2023). Furthermore, bacteria can adsorb heavy toxic metals, such as Pb, Hg, Zn, Cd and Cu, on their cell wall (Priyadarshanee and Das 2021). Bacterial biosorption is regarded as the most reliable and efficient method for the bioremediation of toxic heavy metals (Rizvi et al., 2020). Bacterial biosorption is an effective method because of its specificity for certain metal ions and potential application at low concentrations (Tarekegn et al., 2020). Moreover, biosorption is a passive process facilitated by the presence of active chemisorption sites on the cell wall or extracellular polysaccharides (Mosa et al., 2016). The cell wall functional groups are responsible for the physico-chemical interactions with metal ions. The physical interaction mechanisms involve electrostatic interactions while chemical interaction involve ion exchange displacement of metal cations. Biosorption can be constrained by low nutrient availability in the soil, and this consequently affects the bioremediation efficiency of bacteria (Qin et al., 2013). Biostimulation is the addition of limiting nutrients to promote sufficient biomass growth and optimal biosorption (Priyadarshanee and Das 2021). Biostimulation, using organic solids or synthetic fertiliser, enhances the bioremediation efficiency and accelerates the process (Roy et al., 2011). The use of organic fertilizer and agricultural manure can, however, result in environmental pollution through nutrient runoff into the ecosystem and water bodies (Litskas et al., 2023). Bacterial biosorption is an attractive option for soil bioremediation however, the use of synthetic fertilizer and manure to support biomass growth is economically and environmentally unsustainable. This necessitates the prospection of alternative organic and renewable biostimulants for application in the bioremediation of contaminated soil. Garden waste is an abundant renewable resource and can be valorised into organic compost which is a rich source of carbon and nitrogen. 733 This approach will alleviate pressure from landfills and does not pose threat to food and energy security. Therefore, this study investigated the feasibility of using organic compost, as a sustainable biostimulant, in the bioremediation of lead (Pb) contaminated soil using a bacterial endophyte, Bacillus sp. strain MHSD_36, isolated from the medicinal plan Solanum nigrum. 2. Materials and Method 2.1 Bacterial strains maintenance and growth Routine culture maintenance for the Bacillus sp. strain MHSD_36, isolated from Solanum nigrum, was done by plating a 30% glycerol stock of the bacterial culture on nutrient agar and incubation for 24 h at 28°C. The bacterial culture was grown on nutrient broth (NB) at 30°C, 150 rpm for 24 h. 2.2 Pb biosorption The Bacterial culture grown on NB was harvested and washed with distilled water by centrifugation at 2500g for 10 mins at 4oC. The cells were resuspended in fresh media with Pb (10 mg/L) and without Pb and incubated for 24 h and subsequently centrifuged at 2500g and 4oC for 15 min. The cells were washed twice with 0.03 mol/L Tris buffer containing 2.5 X 10-3 mol/L EDTA, pH 8.0 and resuspended in the buffer. Lysozyme was added to a final concentration of 200 mg/mL and incubated for 30 min at 25oC for the spheroplast preparation. This was followed with centrifugation at 3500 rpm for 15 min and resuspension in 0.03 mol/L Tris buffer containing 3 x 10- 3 mol/L EDTA, pH 8, for the spheroplasts collection. The periplasmic fluid was contained in the supernatant. The spheroplasts were disrupted through the vibronic ultrasonic processor, followed by centrifugation at 3000 rpm for 15 min to enable the removal of debris and unbroken cells. The resulting supernatant consisting of membrane and cytoplasmic fractions was centrifuged at 3500 rpm for 30 min. The pellet consisted of both outer and inner membrane envelopes. The obtained fractions were used for the Pb determination using Inductively coupled plasma optical emission spectroscopy (ICP-OES) after centrifugation at 4000 rpm for 10 min and filter sterilization with a 0.45 µm syringe filter. The % Pb recovered from the different fractions was calculated according to equation 1; % 𝑃𝑏 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 = 𝑃𝑏 ( 𝑚𝑔 𝐿 ) 𝐼𝑛𝑖𝑡𝑎𝑙 𝑃𝑏 ( 𝑚𝑔 𝐿 ) 𝑥 100 (1) 2.3 Determining the impacts of carbon and nitrogen source on Pb biosorption Batch experiments were conducted in 50 ml Erlenmeyer flasks containing 20 mL of nutrients (glucose or peptone) prepared according to the central composite design (CCD). The experiments were carried out with 150 mg/L of Pb, inoculated with 1% v/v cells grown to late exponential phase, and incubated for 48 h at 30oC with shaking at 150 rpm. The culture broth was subsequently centrifuged at 4000 rpm for 10 min, filter sterilized with a 0.45 µm syringe filter and the residual Pb determined using ICP-OES. The % Pb removal was calculated according to equation 2; % 𝑃𝑏 𝑟𝑒𝑚𝑜𝑣𝑎𝑙 = 𝑅𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑃𝑏 ( 𝑚𝑔 𝐿 ) 𝐼𝑛𝑖𝑡𝑎𝑙 𝑃𝑏 ( 𝑚𝑔 𝐿 ) 𝑥 100 (2) 2.4 Soil remediation with compost biostimulation Plastic pots containing 1kg of soil contaminated with Pb (300 mg/Kg) were used for the soil remediation experiments. Compost (40% w/w), obtained from a local nursery, was added as a biostimulant. A compost control was included in the experimental set-up. 100 ml of bacterial culture suspension with a concentration of 1 x 106 cfu/ml was added to the pots. The pots were left standing at room temperature, water was sprayed every second day to keep the soil moist and the soil mixed weekly to ensure sufficient aeration. The experiments were performed in triplicates and terminated after 30 days and the residual Pb in the soil determined with mass spectrometry. 2.5 Experimental design A five-coded levels central composite design (CCD) was used to determine the impact of the carbon (glucose) and nitrogen (peptone) on Pb biosorption. The experiment had 13 runs including 5 center points. The factors were used with the following range, glucose (0.68, 1.0, 1.75, 2.5, 2.81 g/L), and peptone (0.17, 1.0, 3.0, 5.0, 5.8 g/L). The general formula for the response is shown in the equation below: 𝑦i = 𝛽0 + ∑ 𝛽i n i =1 𝑥i + ∑ 𝛽ii𝑥i 2n i=1 + ∑ 𝛽ij𝑥i𝑥j + 𝜖i