Evaluation of Improved Bioremediation Strategy for the Treatment of Abattoir Wastewater using Bacillus licheniformis ZUL012

: The abattoir generates a large volume and variety of biowastes, posing a high risk of environmental contamination, disease outbreaks, and contaminated food. The purpose of this research was to characterize and remediate abattoir wastewater (Aww). The physicochemical characterization of the Aww revealed high level of pollution which served as a baseline for monitoring treatment efficacy. The B. licheniformis ZUL012 isolated from textile wastewater was primed with H 2 O 2 and used to remediate Aww waste water in the current study. This study revealed Aww high pollution level which necessitated a need for the Treatment with this bacterium resulted in a significant decrease in some of the waste water parameters tested. The induced cell reduced the parameters to 155 41 mg/L, 75 mg/L, 454 mg/L, 1750 mg/L, and 1000 mg/L, whereas the naive cell reduced them to 375 mg/L, 776 mg/L, and 3122 mg/L, respectively. This equates to an average reduction of 95 percent (COD), 95 percent (BOD5), 77 percent (TOC), and 71% (TDS) compared to raw wastewater. These novel strategies show that

Abattoirs use a lot of water in their processing operations (slaughtering and cleaning), which results in a lot of wastes (Bustillo-Lecompte et al., 2016). The slaughterhouse generates a large amount and a diverse range of biowastes, posing a high risk of contamination to the environment, disease outbreaks, and unsafe food safety (Al-Gheethi et al., 2021). The wastewater and solid wastes from the abattoirs are directly disposed into bodies of water and lands adjacent to the abattoirs, posing a significant ecological biohazard (Fearon et al. 2014). This is because there are insufficient waste management and treatment facilities. However, the composition of abattoir wastewater (AWW) varies greatly depending on the various industrial processes and water requirements (Bustillo-Lecompte et al., 2015). Meat processing effluents are recognized as harmful and threatening due to the complex composition of fibers, proteins, fats, and high organic content, as well as the pathogenic risk from fecal bacteria and potentially infectious carcasses (Chang et al., 2010;Bustillo-Lecompte et al., 2015;Bustillo-Lecompte and Mehrvar, 2017). In addition, wastewater from slaughterhouses contains a number of toxic materials, including nitrate, detergents, surfactants, and chloric anions, which are classified as emerging contaminants (Latiffi et al. 2021). As a result, it necessitates specialized treatment for a safe and long-term release into the environment (Johns et al., 1995;Bustillo-Lecompte and Mehrvar, 2015). At the same time, inadequate disposal of slaughterhouse biowastes (SHbiowastes) has resulted in harmful algal blooms in surface water (Arvanitoyannis and Kassaveti 2008) and groundwater contamination from antibiotics used in poultry production (Alam et al. 2019), raising safety concerns. Oller et al. (2011) contend that treating recalcitrant, toxic effluent with a single universally applicable end-of-pipe solution is unrealistic, and they advocate a combination of different techniques for economically feasible degradation. Increased oxidative stress improves environmental contaminant elimination and represents a promising strategy in environmental bioremediation by regulating microbial metabolism in oxidoreductase expression (Bains et al., 2019;Liu et al., 2020). Induced oxidative stress increased the production of highly active and efficient oxidoreductases and hydrolases (cytochrome P450, lignin, and horseradish peroxidase) (betaglucosidase). This enzyme family, some of which may also degrade hazardous compounds, is a method for reducing organic micropollutants (Khatoon et al., 2017). The advantages of the Chemically mediated bioremediation are the high efficiency, simplicity, the lack of residues and capacity to treat many different compounds (Hodaifa et al., 2013), at short residence times (Martins and Quinta-Ferreira,2011) and a cost effective process (Mert et al., 2010). The ease of implementation, low cost, and potential to degrade a wide range of organic micropollutants and other pollutants of concern suggest that this mechanism should be researched and exploited for its treatment potential. The objective of this paper is to evaluate physicochemical and Bacteriological Characterization and Treatment of Abattoir Wastewater using Bacillus licheniformis ZUL012

Study Area:
The study site was Bukola Saraki Abattoir located along Sobi road in Ilorin west Local Government Area of Kwara State, Nigeria. The abattoir is a Large-scale business enterprises and it is managed by an Association of butchers and under the supervision of health workers from Ministry of Livestock and Fishery, Ilorin, Kwara State. The slaughtering area is fenced with sand concrete blocks while the floor is made of concrete slab. Normal abattoir operations are carried out from Monday to Sunday. The abattoir is provided with well water but has no slaughter gadgets, cold room and waste treatment facilities. However, there are drainages and channels through which the wastewater leaves the slaughter hall and finally empties into a river around the abattoir.
Collection of Wastewater Samples: Wastewater samples were collected from the abattoir with a sterilized 5-liter can. The Aww was collected aseptically from part of the wastewater running off the drainage system just as it was leaving the slaughter pavements. Samples were transported to the laboratory for the analysis.

Microorganisms:
The bacterium Bacillus licheniformis ZUL012 used in the present study was isolated previously from local textile effluent and soil contaminated with dye effluent obtained from local textile industries in Ilorin (Ajao and Awe, 2018). The Genbank accession is MH411110.

Physicochemical Analyses of Abattoir Waste:
The method of Kumar et al. (2022) was adopted for the determination of the physicochemical parameters of the AWW, such as BOD5, COD, TOC, TDS, EC, pH, phosphate, TN, and TSS as per standard protocol (APHA-AWWA-WPCF, 2017).

Bacteriological Examination of Abattoir Wastewater:
The method of Ajao et al. (2021) was used for the isolation of bacterial contaminants in abattoir wastewater. Membrane Filtration MethodThe membrane filter with a 0.45 m pore size was used to filter 100 mL of water samples.The membranes were aseptically placed on the different agar media. Total coliforms and faecal coliforms counts were enumerated with the use of MarConkey agar and Eosin methylene blue (EMB) agar, incubated at 37 °C and 44.5 °C, respectively. The colonies were enumerated, characterized, and recorded. Their counts were expressed in cfu/100 mL Preparation of Cells Extracts: The pretreated Bacillus licheniformis ZUL012 was grown in LB broth at 37 °C for 18 h. The bacterial cells were harvested by centrifugation (6000 g, 10 min, 4 °C), washed twice with deionised water and re-suspended in deionised water. The density of cell pellet was adjusted to OD600 equal to 0.111 with the inoculum size of approximately 1.5 × 10 8 CFU/mL. Subsequently used as inoculums.
Priming of the B. licheniformis ZUL012 with 0.1 mM H2O2:B. licheniformis ZUL012 pretreated cells with a non-lethal concentration (100 µm of H202) to elicit oxidative stress was demonstrated using method of Rodrı´guez-Rojas et al. (2020) with modification. Bacillus licheniformis strain ZUL012 (1.5 x 10 8 CFU/ml) was exposed (stimulus) to 0.1 mM H2O2 for 30 minutes at 37˚C in an orbital shaker at 100 rpm.
The H2O2 was removed by centrifugation at 4 000 x g for 10 minutes and cells were allowed to recover for 90 minutes, keeping the cell density constant by removing the required volume and replacing it with the appropriate amount of fresh pre-warmed-LB (37˚C) every 30 minutes.
The trigger (1 mM H2O2) was added 90 minutes after removal of the stimulus. The challenge lasted for 30 minutes. The cultures was diluted and plated to AJAO, AT; YUSUF-SALIHU, BO determine cell viability. Non-treated cells were used as control.
Coagulation Process: The method of Upadhyay and Mistry (2012) was followed for the coagulation and flocculation process as follows. About 500ml of abattoir wastewater was collected in the Erlenmeyer flasks 1litre size. Optimized dosage of Al2SO4 was added .pH of sample was adjusted to 8.5 ± 0.2 with use of lime. Mixing at 120 rpm for 30 mins using magnetic stirrer. Then the wastewater was allowed to stand still for 30mins to settle out flocs. After 30mins, supernatant was carefully transferred to the experimental flasks.
Bioremediation Process: The bioremediation process was performed in Erlenmeyer flasks (250 mL) containing 90 mL of autoclaved Abattoir wastewater amended with 0.05 % (v/v) as a source of nutrients. pH was adjusted to 6.8 ± 0.2 using NaOH and HCl 0.1 M solutions. About 1.5ml of PBS (0.02 mM), 1ml of FESO4 (0.75mM) and 1ml of H2O2 (0.01%) was added. Ten percent of the bacterial suspension at the exponential growth phase with the cell density of (OD600 nm =1.11 which correspond to 1.5 × 10 8 cfu/ml were used as inoculums followed by incubation at 30 ± 1 °C and 120 rpm for 10 days. Physicochemical Parameters were determined at every 24 hours intervals to monitor the progress of bioremediation. Statistical Analysis: The data obtained from the experiments were analyzed and expressed as mean and standard deviation. The percentage change was calculated between the controls and experimental for the various experiment. Table 1 shows the results of the physicochemical characteristics of the AWW, which indicate a high pollution load based on the values obtained in all of the measured parameters. This study revealed a high level of organic strength. After the coagulation process, the pH of abattoir wastewater rises from 6.9 to 8.6. All of the measured parameters are on the high side, which could have environmental and public health consequences. The high values obtained in this study for the measured parameters imply that abattoir wastes have a high pollution strength.BOD5 values are widely used to assess the effects of pollution. It represents the amount of putrescible organic matter in water (Kumar et al., 2011).A low BOD content indicates good quality water, whereas a high BOD content indicates polluted water. The BOD5 and COD values obtained were perfectly consistent with the submission of Osuide et al. (2010), who proposed the correlation between BOD5 and COD. In effluent studies, BOD5 and COD are well correlated parameters, and their relative amount depends on the nature of the effluent. COD roughly equals BOD5 in effluents containing largely biodegradable pollutants, whereas COD exceeds BOD5 in effluents containing largely non-biodegradable pollutants. This waste contains a high concentration of TDS. The COD, BOD5, TOC, and TDS values indicate a high level of organic and inorganic matter pollution. The coagulation process reduced the mean values of the pollution indicators determined: BOD5, COD, TOC, and TDS, while no noticeable changes occurred in total phosphorus or total nitrogen. There were high levels of coliforms and total heterotophic bacteria counts. Their population was drastically reduced as a result of the coagulation process. Phosphate and nitrate are two of the most common compounds found in abattoir effluent. This effluent had higher levels of phosphate and nitrate compounds. This disparity could be attributed to the effluents' high fecal content. According to Rodier (2009), before being discharged into the aquatic environment, wastewater samples must contain less than 50 mg/l of nitrates and 0.5 mg/l of phosphate. The river will become eutrophic as a result of high phosphate levels. Blood also contributes significantly to the nitrogen content, while phosphorus is derived from stomach contents in the effluent. In the study of Ijah (2016); Elemile et al., a similar pattern of results was observed (2019).   (Seema et al., 2009). Table 3 shows the result of treatment which was done for five days at interval of 24 hours. The Initial values of organic Pollutant index, Biochemical oxygen demand (BOD5), Chemical oxygen demand (COD), and Total Organic carbon (TOC) and Total Dissolve Solid were 1420 mg/L, 3263 mg/L, 2000 mg/L and 5978 mg/L respectively before the commencement of the bioremediation process. The efficiency of organic micropollutants biotransformation is linked to environmental conditions that enhance the expression of oxidoreductases by microbes (Alneyadi et al., 2018). To improve the biodegradability of microorganisms is to induce microorganisms to produce degradative enzymes (Fischer and Majewsky, 2014). This study demonstrated an evidence that the increase of oxidative stress artificially could improve the bioremediation of abattoir wastewater derived from B. licheniformis ZUL012. The hypothesis that can explain the high degradation observed in this study is that the priming of the bacterial cell induces oxidative stress which elicit gene expression that enhanced the bioremediation of the abattoir wastewater as measured in terms of the reduction in the organic pollutant strength of the abattoir wastewater such as COD, BOD5, TOC and TDS by both the induced and naive cell. The induced cell reduced the parameters down to 155 ± 41 mg/L, 75 mg/L, 454mg/L and 1750 mg/L and 1000 mg/L respectively while naive cell reduced them to 375 mg/L, 776 mg/L and 3122 mg/L respectively  This corresponds to an average reduction of 95 % (COD), 95 % (BOD5), 77% (TOC)and 71% (TDS), relative to the raw wastewater while naive cell provides Average reductions of 69 % (COD), 74 % (BOD5), 61 % (TOC)and 48 % (TDS). The reduction percentages reported in this study are higher than those previously reported in the literature. Overall, the combined process was sufficient to meet discharge requirements without further treatment for the measured parameters (COD, BOD5, TOC, and TDS). This study confirmed the induction of the oxidative enzyme gene expression mediated by H2O2. This pattern of observation has been shown in previous works who reported that the regulation of microbial metabolism in oxidoreductase expression consequentently synthesis antioxidative enzymes such as peroxidases and oxidoreductases to protect against such oxidative stress, these enzymes, some of which are also capable of degrading toxic substances and facilitates the removal of environmental pollutants (Bains et al., 2019;Liu et al., 2020).

Conclusion:
This study showed that the Aww was grossly polluted with high organic strength and bacterial pathogens. Iron-H2O2-modified wastewater mediated with the B. licheniformis ZUL012. The results showed that approach was the most efficient to remove BOD5, COD, TOC and TDS from slaughterhouse wastewater and also ensured dual advantages of a shorter reactiontime and cost effective process.