Bioremediation Potential of Indigenous Bacteria from Piggery Effluents in Owo, Nigeria

Abstract
Piggery wastes can be reservoirs of heavy metals and high microbial loads that pose environmental and public health risks. This study evaluated the bioremediation potential of indigenous bacteria isolated from piggery effluents collected from six farms in Owo local government area, Nigeria. Bacterial isolates (Escherichia coli, Klebsiella sp., Pseudomonas sp. and Staphylococcus sp.) were recovered and characterized by standard cultural, morphological and biochemical tests. Isolates were incubated in nutrient broth amended with target metal salts and metal concentrations were monitored weekly for 28 days by atomic absorption spectrophotometry. Pseudomonas sp. showed the greatest reduction in selected heavy metals, while Escherichia coli demonstrated notable reduction of zinc. These findings indicate that indigenous piggery bacteria particularly Pseudomonas spp. have measurable potential for laboratory scale bioremediation of metal-contaminated wastes. We recommend follow-up studies using quantitative molecular identification, controlled microcosm/mesocosm trials and assessment of safety (e.g., antibiotic resistance profiling) before field application.
Keywords
Bioremediation; piggery effluent; Pseudomonas; heavy metals; Nigeria; biosorption

INTRODUCTION
Livestock is very important in contributing to the sustainability of agricultural systems as an integral part of traditional farming system by using crop residues and other feeds which are not utilized by humans and converting them into milk and meat (Martinez et al., 2009). Manure from different livestock species can replace a good amount of mineral fertilizer as plant nutrients, provided it is recognized and used accordingly as a valuable resource (Martinez et al., 2009). However, at the same time animal manure can be a source of air pollution as well as a threat to aquifers and surface water. Commercial pig production is rapidly growing in most parts of the world with a trend towards larger production units utilizing modern production technologies such as modern housing, improved feeding and better breeding methods andthis type of trends influences the risk of water pollution, air pollution as well as soil pollution (Gerber et al., 2005).
Landfills are thought to account for roughly 95% of the solid waste produced globally and are widely regarded as the most cost-effective method of disposing of waste (Scott et al., 2005). According to Hoornweg and Bhada-Tata's (2012), World Bank reports the total amount of waste produced by urban centers worldwide is 1,300 million tonnes annually (or 1.2kg per capita per day). Nigeria produces roughly 62 million tonnes of waste annually, or 0.65kg per person per day, more than any other country in sub-Saharan Africa. By 2025, the country's waste output is expected to reach 161.27 million tonnes, or 0.85kg per person per day. In developing countries, landfilling is virtually the only approach of waste management (Rubinos and Spagnoli, 2018). Unfortunately, in major cities of Nigeria, these largely unsegregated and heterogeneous in composition wastes are dumped in open grounds, which are not engineered to prevent serious contamination of the underlying soil, ground and surface waters by toxic materials leached from the wastes (Misra et al., 2018). Furthermore, with the 2012 World Bank report also posting a 5% presence of metals in the wastes generated in Nigeria, several authors have documented the presence of heavy metals like, arsenic, cadmium, chromium, nickel, lead copper, mercury and zinc (Gbadamosi et al., 2018).
Bioremediation has emerged as a new low-technological cheap technology that uses plants and their associated microbial flora for environmental cleanup (Oladipo et al., 2014). It is a biological treatment that offers outstanding environmentally friendly method for remediating heavy metal polluted soil; as it utilizes the ability of the indigenous microorganisms in the soil environment to break down the heavy metals into their harmless components, several studies had been carried out on landfill sites reporting various microorganisms efficient for bioremediation (Koshlaf et al., 2019; Hassan et al., 2020).
Bioremediation is a most efficient, eco-friendly and cost-effective technology for the transformation of contaminants (Sonune, 2021). Biological remediation can be carried out using both plants and microorganism, nonetheless, plants take a longer time to grow and cannot be easily manipulated like the microbes which makes the microbes more preferable (Hussain et al., 2022). In addition, microbes mitigate heavy metals and improve soil fertility and plant development (Chaudhary et al., 2023).

According to Ayilara and Babalola (2023), the rise of urbanization and industrialization, has left the environment exposed to numerous pollutants which are toxic to living things. Pollutants arising from different industrial processes are major sources of pollution to the soil and aquatic environment. Different types and quantities of heavy metals are released during the industrial production process and as effluents after further industrial production. For instance, the wastewater from dye-producing companies is associated with antimony, chromium and mercury. Similarly, untreated pollutants from wastewaters of the agri-food industries disposed into river canals and other water bodies have harmful effects on the environment (AL-Huqail et al., 2022).
In addition, copper, arsenic, mercury, chromium, lead, nickel, cadmium and zinc from the coal industry serve as environmental pollutant (Sun et al., 2019; Fashola et al., 2020). These heavy metals are very toxic to aquatic and terrestrial habitats and their inhabitants, in humans, mercury, cadmium and lead alters the central nervous system, especially in infants, while lead results in liver and kidney dysfunction, cardiovascular diseases, malfunctioning of the reproductive and immune system (Gangola et al., 2018; Ayangbenro and Babalola, 2020). This study therefore aims to evaluate the bioremediation potential of indigenous bacteria on heavy metals in piggery effluents.
MATERIALS AND METHODS
Study sites
The piggery dung was collected from 6 selected farm sites in Owo local government and piggery dung was collected using sterile and clean bottles
Microbial analyses 
 The media were prepared according to manufactures instructions. For enumeration of total colony forming unit,Nutrient agar was employed while for the isolation of enteric bacteria, MacConkey agar (MAC) was used. After serial dilutions were done, 1ml of the last test tube was inoculated into the media prepared using pour plate techniques. After that, the plates were incubated at 37°C for 24 h. Following incubation, colonies on nutrient agar plates were counted and calculated by multiplying average number of colonies in particular dilution with dilution factors and recorded as colony-forming unit per gram of samples.
Isolation of pure colonies
To isolate pure colony, bacteria that grow on different media were sub-cultured on fresh nutrient agar plates. All the isolates of respective bacteria were identified based on cultural characteristics, morphological characteristics and biochemical tests (Sonune, 2021).
Characterization of isolates
Characterization of the isolates was done using phenotypic methods including Gram staining, biochemical and sugar fermentation tests.
Biochemical Tests
The isolates were differentiated by carrying out some biochemical tests such as urease, citrate and indoles, catalase, coagulase, oxidase etc.
Heavy metal bioremediation evaluation of the isolates
Nutrient broth (95 ml) was prepared and 5 g of each metal salt was incorporated to make 5 % concentration. This mixture was then sterilized in the autoclave for 15 minutes. Thereafter, Pure isolates were inoculated into sterile nutrient broth amended with individual metal salt. Control tube was left without inoculation. The set up was incubated for 28 days at 35 oC. The heavy metal present in the tubes were monitored at weekly interval using Atomic Absorption Spectrophotometer (Omoni et al., 2015).
Statistical Analysis
Data are expressed as mean ± standard error of triplicate determinations. Two-way ANOVA followed by Duncan's multiple range test was used to compare treatments; significance was accepted at p ≤ 0.05.
 RESULTS AND DISCUSSION
The table.1 below shows the total colony-forming unit of samples of each location respectively. It was observed that the load ranged between 3.2 and 4.1 x 108cfu/g. The high number of the bacteria in the piggery effluent is not unexpected as the wastes from animal production centers are known to contain high level of organisms which may be linked with high nutrient density in these wastes which supports the growth and proliferation of the cells.
The table 2 revealed that only four types of bacteria were identified from the waste, these include Escherichia coli, Klebsiella sp., Pseudomonas sp. and Staphylococcus sp., these had been reported earlier by other researchers. Their presence are not unusual because they are part of normal floral of the intestine of the warm blooded animals.
This low types despite the high level of count may be linked with the use of antibiotics by the farmers. From the personal observations and interview held with the farmers, it was revealed that there is a high level of antibiotics consumption for the prevention and treatment of different diseases among the pigs.
Table 1: Bacterial counton the piggery effluents (cfu/g)
LOCATION	1	2	3	4	5	6	
COUNT	3.5×108	3.2×108	4.0×108	3.3×108	3.8×108	4.1×108	

The four Figures (1–4) show changes in heavy-metal concentrations (Pb, Cd, Ni, Zn; ppm) in broth cultures inoculated with isolates obtained from piggery waste (Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Staphylococcus aureus) and an uninoculated control over a 4 week incubation at growth temperature. Overall, most isolates reduced metal concentrations relative to the control; P. aeruginosa produced the largest and most consistent reductions across all tested metals, E. coli showed pronounced removal of Zn, while S. aureus showed little or no removal. These results indicate potential for microbial bioremediation of heavy-metal contaminated environments, with Pseudomonas as a leading candidate for further development.
The concentrations of lead (Pb), cadmium (Cd), nickel (Ni) and zinc (Zn) in broth cultures changed markedly when inoculated with the isolates obtained from piggery wastes (Figures 1–4). Across all four metals, Pseudomonas aeruginosa produced the largest and most consistent reductions in metal concentration over the 3–4 week incubation period relative to the uninoculated control (Figures 1–4). Escherichia coli showed notable removal of Zn (Figure 4) but was less effective than P. aeruginosa for Pb, Cd and Ni. Klebsiella sp. produced moderate reductions for several metals, whereas Staphylococcus aureus exhibited little or no change in metal concentration relative to the control over the experiment. 

Table 2: Biochemical Test Results
ORG		BIOCHEMICAL TEST RESULTS	
	Gram	OXIDASE	COAGULASE	CATALASE	UREASE	MOTILITY	CITRATE	GLUCOSE	LACTOSE	MALTOSE	INDOLE	
Klebsiella sp.	-	-	-	+	+	-	+	+	+	+	-	
Pseudomonassp	-	+	-	+	-	+	-	-	+	-	-	
Staphylococcussp	+	-	+	+	-	-	-	-	-	-	-	
E.coli	-	-	-	-	-	+	-	+	+	-	+	

Table .3: Occurrence of the isolates  ineach Location Samples
LOCATIONS	
ORGANISM	1	2	3	4	5	6	
E. COLI	+	+	+	+	+	-	
KLEBSIELLA	+	+	-	-	+	-	
PSEUDOMONAS	+	+	-	-	+	-	
STAPHYLOCOCCUS	+	-	+	+	+	+	

Figure 1. Reduction of lead (Pb) concentration (ppm) in broth over 4 weeks by bacterial isolates from piggery waste. Bars show mean metal concentration (ppm) measured weekly for cultures inoculated with E. coli, Klebsiella sp., P. aeruginosa, S. aureus, and an uninoculated control (n = X biological replicates; error bars = ± SD). P. aeruginosa achieved the greatest Pb removal by week 4.

Figure 2. Cadmium (Cd) concentration (ppm) in broth over 4 weeks for the same isolates and control. P. aeruginosa reduced Cd substantially; S. aureus showed minimal change. Error bars represent standard deviation

Figure 3: The bioremediation effect of the bacterial isolated from piggery wastes on Ni concentration in 4 weeks

Figure 4: The bioremediation effect of the bacterial isolated from piggery wastes on Zn concentration in 4 weeks

The health risk associated with potential poisoning due to heavy metal pollution ranging from consumption of plants with bio-accumulated metals and animals (aquatic life) has increase the need for environmental clean-up from heavy metal pollution. The results obtained from this study suggest that the two organisms may be employed in the clean-up of environments contaminated by these metals. Similar observation has been made by Deip et al. (2018) who reported bioremediation potential of bacteria on heavy metals in oil-polluted soils. These trends indicate species-specific differences in metal removal capacity under the tested conditions.
The ability of microbes especially Pseudomonas sp. to bioremediate heavy metals have been reported to be determined by different factors, which include the total metal ion concentration, redox potential, chemical forms of the metals, competition among microbes, pH, temperature, soil structure, presence of oxygen, moisture content, nature of the soil and the solubility of the heavy metal (Khan et al., 2018; MedfuTarekegn et al., 2020).
The observed variation in heavy metal reduction among isolates likely reflects species-specific mechanisms of metal uptake, biosorption to cell surfaces, intracellular sequestration, and/or metal transformation. P. aeruginosa’s superior performance across Pb, Cd, Ni and Zn is consistent with its known metabolic versatility and the presence of metal resistance and efflux systems, extracellular polymeric substances that bind metals, and siderophore production that can chelate metal ions (Albelda-Berenguer et al., 2019). The strong Zn removal by E. coli suggests a higher affinity or capacity for Zn biosorption in the strain tested. Minimal removal by S. aureus may reflect absence or low expression of metal binding/sequestration pathways under the culture conditions used. Environmental and experimental factors — total metal concentration, pH, redox potential, culture age, incubation temperature, ionic strength, and presence of competing ions — also influence observed removal efficiencies and could explain differences between isolates (Xiao et al., 2019; Bhandari et al., 2023). Together, these results support further development of Pseudomonas-based strategies for bioremediation of metal-contaminated waste and soils; however, in situ effectiveness must be validated because laboratory broth systems do not replicate the complexity of soils/sediments or plant rhizospheres (Xia et al., 2019; Alvarez et al., 2020).
Our results indicate that indigenous bacteria from piggery effluents can decrease dissolved concentrations of selected heavy metals in laboratory broth assays. The superior performance of Pseudomonas spp. is consistent with numerous contemporary reports that highlight the genus's versatility in metal resistance and remediation via mechanisms such as biosorption to cell walls, secretion of extracellular polymeric substances (EPS), siderophore production, and intracellular sequestration (Weyens et al., 2009). Recent reviews emphasize EPS and siderophores as major facilitators of microbial heavy-metal immobilization and tolerance (Arstol and Hohmann-Marriott, 2019). Pseudomonas strains isolated from agricultural wastes and composts have been shown experimentally to tolerate and remove Cd and Zn, supporting our observation that Pseudomonas effectively reduced these metals in vitro. The limited activity observed for Staphylococcus in our assays may reflect lower intrinsic metal-binding capacity, absence of effective EPS-mediated sequestration, or assay-specific factors (metal speciation, pH, ionic strength) (Zhang et al., 2010; Zacharia, 2019). Microbial removal efficiency depends strongly on environmental parameters; thus, laboratory broth results should be interpreted as proof-of-concept rather than direct field-scale efficacy. Recent reviews recommend follow-up microcosm and mesocosm experiments under controlled but environmentally realistic conditions before field deployment (Cao et al., 2020). Finally, piggery effluents may also contain antibiotics and antibiotic-resistant bacteria that pose biosafety concerns for bioaugmentation strategies; contemporary guidance therefore advises concurrent profiling of antibiotic resistance and pathogenicity markers prior to environmental release of isolates. 

CONCLUSION 
It may be concluded from this study that Pseudomonas sp., Klebsiella sp, E. coli, and Staphylococcus sp. were associated with the effluent of piggery in Owo. Moreover, Pseudomonas sp. had higher bioremediating activity on the selected heavy metals. However, there is need to integrate possible stronger policies on environmental protection such as the necessity to include new emerging pollutants such as antibiotics, endocrine disrupters and antibiotic resistant pathogens.

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