Constructing a microbial diversity profile for red meat abattoir effluent

Abstract

Abattoirs are critical components of the global food supply chain as they provide essential resources for populations worldwide. However, the environmental footprint of these facilities, particularly through the generation of effluent, poses significant challenges to sustainable water management and environmental conservation. Abattoir effluent is distinguished by its high biological load, which refers to the concentration of organic materials including stomach content, blood, and large amounts of fats, oils, and grease (FOG). While the physicochemical properties of abattoir effluent have been extensively studied, little is known about the microbial diversity within the effluent. This knowledge gap represents a critical barrier to understanding the full environmental and health implications of abattoir effluent discharge, especially in regions where it is released into municipal sewer systems without adequate pre-treatment. Understanding the microbial constituents of abattoir effluent can inform targeted interventions to remove harmful pathogens and reduce the organic load, thereby alleviating the burden on municipal wastewater treatment systems. Ensuring compliance with existing municipal by-laws and regulatory standards that govern effluent discharge is also important in alleviating this burden. This study aimed to construct a detailed profile of the microbial diversity present in red meat abattoir effluent by focusing on both genomic and metabolic capabilities as well as physicochemical compliance with local regulatory standards. The sampling site was a high throughput red meat abattoir in Bloemfontein, Free State. An abattoir is considered to have a high throughput if it processes more than 20 units daily, with the unit count based on the type of animal slaughtered. Red meat abattoirs are facilities where various animals such as cattle, sheep, pigs, or horses are slaughtered. Daily sampling was performed for 10 consecutive days by collecting composite samples from the pipe through which effluent was discharged into the sewer system. Physical and chemical parameters were considered significant when characterising the abattoir effluent environment were analysed. Community Level Physiological Profiles (CLPP) were constructed using BioLog EcoPlate sole-carbon substrate utilisation analysis while culturable bacteria and fungi were quantified. Enumerations of total microbial load, Escherichia coli (E. coli), Pseudomonas aeruginosa, Enterobacter aerogenes, and yeast and mould were performed using rich (Nutrient) and/or selective agars (Harlequin®, Nutrient Agar and Rose Bengal Chloramphenicol). The genetic diversity was determined through 16S- and ITS-targeted metagenomic sequencing on the Illumina MiSeq platform. Physicochemical parameters indicated 37.5% non-compliance with the Mangaung effluent discharge limits. Non-compliance was attributed to high levels of solids, mostly volatile, high chemical oxygen demand (COD) due to the organic loads as well as high alkalinity levels. The environment had a neutral pH and low EC levels that were within the Mangaung effluent discharge limits while sufficient levels of dissolved oxygen (DO) were also detected. The temperature profile was consistent throughout ranging between 24°C-28ºC. Nutrients such as phosphate, total organic carbon (TOC), sulphate, and chloride were also detected at levels that were within the discharge limits. As expected, high microbial loads were noted, with bacteria being the more dominant microorganisms compared to fungi. Bacterial coliforms, Pseudomonas aeruginosa (8.04×105 CFU.mL-1), Enterobacter aerogenes (1.18×105 CFU.mL-1) and E. coli (2.78×104 CFU.mL-1) were present. Low counts of yeast (5.32×103 CFU.mL-1) and mould (7.10×101 CFU.mL-1) were observed. CLPP displayed an abundant, diverse, and functionally active community. Simpson index (0.962) measurements displayed diversity, the evenness index (0.97) depicted the presence of an abundance of species, and the average well colour development symbolised a functionally active consortium. The CLPP showed consistency amongst all the sample days. The relative abundance of metabolic activities was related to polymer, carbohydrate, and amino acid category metabolism. Glycogen and L-asparagine were the most utilised substrates while 2-hydroxy benzoic acid and α-ketobutyric acid were not utilised. The taxonomic bacterial and fungal biodiversity showed a broad similarity. Bacterial diversity showed consistent dominance of Bacteroidetes, Firmicutes, and Proteobacteria phyla. Genus richness varied in dominance distribution, with Acinetobacter, Bacteroides, and Rikenellaceae_RC9_gut_group being the most abundant genera with the dominance varying for each sample. Dominant fungal phyla were either Ascomycota or Neocallimastigomycota. Basidiomycota appeared in small, varied levels for each sample. The distinctive presence of Mucoromycota was observed on days when pigs were slaughtered, which deviated from the normative microbial composition encountered during the other sampling days, thus signifying an exception within the microbial community dynamics. The fungal community was dominated by a particular genus in each sample, namely Neocallimastix, Caecomyces, and Ciliophora respectively. Both the culture-dependent and culture-independent analyses of the microbial content yielded noteworthy findings that indicated an exceptional degree of uniformity across all sample days. The calculated similarity rate ranging from 96-98% highlighted a distinct resemblance in microbial diversity among the samples. The effluent consistently exhibited a comparable microbial content except for sample day 6 phylum discrepancies, which accounted for the 2-4% difference in the similarity rate calculated. These findings strongly indicate a substantial overlap in microbiomes between the meat, stomach content, and hide of different animals, suggesting a commonality in the microbial species present in the effluent. Given the limited information available on the microbial diversity of abattoir effluent, this research filled a significant gap in the environmental science and public health domain as it was able to contribute significant knowledge of the complex microbial ecosystems within abattoir effluent. The findings could pave the way for innovative treatment strategies using indigenous microbes and may thus contribute to more sustainable abattoir effluent discharge practices.