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Communication

A Snapshot of the Influent and Effluent Bacterial Populations in a Wastewater Treatment Plant in the North-West Province, South Africa

by
Stenly Makuwa
1,
Ezekiel Green
1,
Elvis Fosso-Kankeu
2,
Victor Moroaswi
3 and
Matsobane Tlou
3,*
1
Department of Biotechnology, University of Johannesburg, Doornfontein 2028, South Africa
2
Department of Mining Engineering, College of Science Engineering and Technology, Florida Science Campus, University of South Africa, Roodepoort 1709, South Africa
3
Department of Biochemistry, School of Physical and Chemical Sciences at the North-West University, Mafikeng 2790, South Africa
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2023, 3(3), 764-773; https://doi.org/10.3390/applmicrobiol3030053
Submission received: 27 May 2023 / Revised: 9 July 2023 / Accepted: 9 July 2023 / Published: 13 July 2023
Browse Figure
Review Reports Versions Notes

Abstract

:
Wastewater treatment plants receive influent wastewater that is contaminated with bacterial pathogens which may be released into the environment if the plant effluent is inadequately treated. In this study, next-generation sequencing was used to perform a 16S rDNA-based survey of bacterial populations in the influent and effluent from a treatment facility in the North-West Province (SA). In total, 3638 and 3872 effective DNA reads were obtained for the influent and effluent, respectively. Sequence analysis revealed the detection of a diverse bacterial constituency in both the influent and effluent samples. The phyla: Proteobacteria (49.82% and 52.04%), Firmicutes (14.06% and 13.14%) and Actinobacteria (5.00% and 9.99%) were found to be taxonomically abundant in the influent and effluent, respectively. This translated to the detection of biological treatment-, fecal coliform-, and disease-associated bacterial groups that are classified under the following genera: Escherichia spp., Serratia spp., Aeromonas spp., Legionella spp., Pseudomonas spp., Mycobacterium spp., Clostridium spp., Staphylococcus spp. and Streptococcus spp., Comamonas spp., Nitrosomonas spp., Acinetobacter spp., Rhodobacter spp., Paracoccus spp., Hyphomicrobium spp., and Desulfovibrio spp.

1. Introduction

Effluent wastewater refers to treated water that is discharged from a treatment facility which can be used to improve the quantity/volumes of environmental water catchments and hygiene in urban environments [1]. The wastewater treatment plant (WWTP) in this study receives (influent) municipal domestic sewage and wastewater that is heavily influenced by industrial water use [2]. In general, there is an exponential increase in influent pollution loads due to rapid urbanization, industrialization and population growth [3,4]. The combination of limited wastewater treatment infrastructure in various regions worldwide along with other factors known to impact the efficiency of treatment plants [2,3,5] raises concerns about the potential discharge of pathogens into the environment. Such pathogens have the capacity to trigger a wide range of waterborne diseases. This is particularly concerning in the context of a country like South Africa (SA) where many communities still rely on surface water obtained directly from streams, rivers and dams [6,7].
In wastewater treatment, chlorine-based disinfection is the method of choice for the inactivation of common bacterial indicator organisms [8,9] and has been reported to be the most effective [10]. The chemical results in the elimination and (or) inactivation of pathogens into non-reproductive forms. However, it is now accepted that chlorine can be ineffective against some microbes, and pathogens capable of repair and re-growth post-disinfection have been reported in effluent wastewater [9,11]. Moreover, the detection in effluent wastewater of high titers of indicator E. coli [2] and the isolation of antibiotic resistant pathogenic strains [10,12] further underscores the potential risks associated with the domestic use of water in the river catchments.
In 2016, Zhi and co-workers [13] reported on the presence of naturalized E. coli strains in municipal wastewater that were adapted to differential survival of the treatment process. The isolates were subjected to a serial stress experiment where nutrient deprivation and osmotic stress followed by chlorine treatment resulted in strains that were 100 times more resistant than a wild-type human fecal strain [13]. Bacterial chlorine resistance is due to a conserved response to DNA damage (SOS response) triggered by oxidative stress [14], while oligotrophic conditions result in quiescent growth and metabolism that may lead to bacteria in a viable but non-cultivable state for chlorine stress adaptations [15,16].
Historically, culturing methods have been widely employed to characterize microbial communities, offering a direct and effective approach. However, it is important to acknowledge that a significant portion of bacteria found in the natural environment cannot be cultured using conventional laboratory media [17,18,19]. This uncultured bacterial diversity is substantial, underscoring the limitations of culture-dependent techniques.
The emergence of next-generation sequencing (NGS) technologies has revolutionized the field by enabling “culture-independent” high-throughput sequencing approaches at a relatively low cost [20]. Pyrosequencing, in particular, has proven to be an effective NGS system, capable of generating over 40,000 high-quality reads with average lengths reaching several hundred base pairs and an average quality above 99% [20,21]. This technology has been successfully employed in studying microbial diversity and abundance in various samples, including marine water [22] and the human distal intestine [23].
By leveraging NGS technologies like pyrosequencing, researchers have been able to overcome the limitations of culture-based methods, allowing for a comprehensive exploration of microbial communities without relying solely on culturable organisms. These sequencing techniques provide a more accurate representation of the true microbial diversity and have greatly enhanced our understanding of complex microbial ecosystems.
In a study conducted by Makuwa et al. [2], the performance of the wastewater treatment plant (WWTP) in question was assessed based on its ability to reduce fecal coliforms and physicochemical indicators in the effluent discharged. However, in the present study, a different approach was adopted by utilizing next-generation sequencing (NGS) technology for a culture-independent analysis of the influent and effluent bacterial populations. This approach aimed to provide a comprehensive understanding of the bacterial communities, including potential pathogens, that are received by and potentially released into the environment from the treatment facility.
By employing NGS technology, this study was able to overcome the limitations of culture-based methods and explore the microbial diversity in a more inclusive manner. The use of NGS allowed for a deeper insight into the bacterial populations, including the detection of potential pathogens, shedding light on the composition and potential risks associated with the treated effluent discharged into the environment. This culture-independent examination provided valuable information about the microbial communities and their dynamics within the WWTP, enhancing our understanding of the treatment process and its potential impact on the environment.

2. Materials and Methods

2.1. Sample Collection and DNA Extraction

The influent and effluent wastewater samples were collected in a sterile manner using 1 L sampling bottles specifically designed for aseptic sampling. Single-time sampling was performed to capture a snapshot of the microbial populations at that moment. To remove larger and coarse particles from the samples, they were initially filtered through a Whatman filter paper #114, which aids in the removal of these undesirable components. Next, the filtrate samples underwent further filtration using a 0.20 µm Supor® membrane filter manufactured by PALL Life Sciences, New York, NY, USA. This step aimed to concentrate the bacterial cells present in the samples. A peristaltic pump was used to facilitate the filtration process. Following the filtration steps, total genomic DNA was extracted from the concentrated bacterial cells using the Soil/Fecal DNA Extraction Kit™ provided by Zymo Research Corporation, Tustin, CA, USA. The DNA extraction protocol provided by the manufacturer was followed to ensure efficient and reliable extraction of the DNA. Once the DNA extraction was completed, the total DNA obtained from the samples was eluted, and its concentration was quantified using a Qubit 3.0 Fluorometer, a device manufactured by Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA.

2.2. DNA Sequencing and Sequence Analysis

Amplification of the 16S rDNA gene was carried out with primer sets 27F (5′-AGAG TTTGATCMTGGC-3′) and 518R (5′-ATTACCGCGGCTGC TGG-3′), as described by García-Moyano et al. [24]. The PCR reactions constituted 25 μL of one Taq 2X Master Mix, 1.5 μL each of the forward and reverse primers at a concentration of 0.2 μM, 2 μL of extracted DNA (50–100 ng μL−1), and 22 μL of nuclease-free water. The thermal profile consisted of an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min, followed by cooling to 4 °C. The PCR products were purified using a DNA Clean and Concentrator Kit (ZYMO RESEARCH, Irvin, CA, USA) and sent for pyrosequencing on the GD-FLX-Titanium series 454 and sequence analysis (Inqaba Biotech, Pretoria, South Africa). Briefly, the PCR products were barcoded with Pacbio M13 barcodes for multiplexing through limited cycle PCR (www.pacb.com (accessed on 25 November 2020). The resulting barcoded amplicons were quantified and pooled with equimolar, and then AMPure PB bead-based purification step was performed. The PacBio SMRTbell library was prepared from the pooled amplicons following the manufacture’s protocol. Sequencing primer annealing and polymerase binding were performed following SMRTlink Link software protocol to prepare the library for sequencing on the PacBio Sequel IIe system. Raw subreads were processed through the SMRTlink (v10.2) Circular Consensus Sequences (CCS) algorithm to produce highly accurate reads (>QV40). These highly accurate reads were processed through DADA2 (https://benjjneb.github.io/dada2/index.html (accessed on 29 November 2020)) and qiime2 (https://docs.qiime2.org/2021.11/ (accessed on 29 November 2020)) for quality control assessment and taxonomic classification, respectively.

3. Results and Discussion

Microbial diversity depends on the type of treatment system applied in a WWTP; therefore, a selected treatment configuration can favor the species’ structure of biomass that supports process stability and efficiency [25]. In this study, we analyzed the bacterial diversity from the influent and effluent samples taken from an activated sludge municipal WWTP in the North-West Province of South Africa. The bacterial taxonomic diversity was obtained upon next-generation sequencing (NGS) analysis of the 16S rDNA amplicon sequences. According to Numberger et al. [1], the taxonomic determination of 16S rDNA genes allows for improved characterization of potential pathogenic taxa. In this study, a total of 3638 and 3872 effective reads were recorded from the influent and effluent samples, respectively. The reads obtained here are significantly lower when compared to the 21,861 and 20,250 effective reads for the activated sludge and digestion sludge, influent and effluent samples, respectively [26]. The DNA or RNA reads are referred to as the number of corresponding base pairs obtained from DNA/RNA fragments through NGS analysis [27,28]. According to Kukurba and Montgomery [29], for any given study, it is important to consider the level of sequencing depth required to answer experimental questions with confidence while efficiently using NGS resources. Kuśmirek et al. [27], stated that the third-generation sequencing techniques make it possible to obtain much longer DNA/RNA reads compared to the NGS technology that was used in this study.
The most represented phyla detected in this study were Proteobacteria (49.82% and 52.04%), Firmicutes (14.06% and 13.14%) and Actinobacteria (5.00% and 9.99%), from the influent and effluent, respectively (Figure 1a). However, the study recorded an increase in the relative proportion of the phyla Proteobacteria and Actinobacteria at the effluent compared to the influent. According to Numberger et al. [1], WWTPs may enrich and release increased bacterial quantity into the environment. Their study demonstrated an increase in relative proportion of Legionella and Leptospira from inflow to effluent. Chlorination can effectively destroy or decrease the abundance of bacteria from the raw water, but not eliminate their DNA from the water. Disinfected samples or samples that contain residual chlorine may contain DNA and dead or damaged biomass and may influence the relative abundance in the populations [30]. The DNA or RNA based methods cannot differentiate between viable and dead bacterial cells or extracellular DNA or RNA [31], and this suggests that chlorination has inconsistent impacts on the genetic material, which is an important reflection for data interpretation.
The predominance of Proteobacteria observed in this study aligns with findings from previous research conducted on effluent samples from various wastewater treatment plants (WWTPs), where this phylum accounted for 21–53% of total bacterial effective sequences [4,25,26,32]. Proteobacteria is a diverse group of Gram-negative bacteria with a wide range of metabolic capabilities [33], and its prevalence raises concerns regarding potential threats to human health.
In the activated sludge process, bacteria are utilized for organic matter degradation and nutrient cycling [34], and Proteobacteria have been recognized for their significant contributions to these processes [35]. In this study, Betaproteobacteria emerged as the most dominant class within the Proteobacteria phylum (Figure 1b). Consistent with existing literature, this class is primarily associated with organic and nutrient removal [25].
Epsilonproteobacteria, the second most dominant group detected in the influent and effluent samples, is commonly found in sewage samples. Many members of Epsilonproteobacteria are known to be either commensal, pathogenic, or free-living organisms [36].
Among the other phyla of notable dominance, Actinobacteria and Firmicutes were observed. These phyla consist of Gram-positive bacteria that are generally recognized for their ability to withstand chlorine disinfection due to their thick cell walls [30]. Within the Firmicutes phylum, Clostridia emerged as the dominant Gram-positive bacterial class, while Actinobacteria represented the dominant class within the Actinobacteria phylum (Figure 1b).
Overall, the results support the notion that Proteobacteria, particularly Betaproteobacteria, play crucial roles in organic matter and nutrient removal within the activated sludge process. The presence of Epsilonproteobacteria, Actinobacteria and Firmicutes further contributes to the microbial diversity and functional dynamics within the wastewater treatment system.
The primary objective of a wastewater treatment facility, as is the case globally, is to ensure that effluents from industrial and domestic activities are safely disposed of without posing harm to human health or the environment. The treatment facility in this study, like many others, utilizes a combination of activated sludge and chlorination processes. Activated sludge employs bacteria for various functions such as nitrification, denitrification and biological removal of excess phosphorus and sulfur, known as biological nutrient removal.
Table 1 demonstrates that bacterial species responsible for nutrient removal were detected in the final effluent. This confirms the influence of the activated sludge process on the microbial populations in the effluent, suggesting that these microorganisms have the potential to survive chlorine disinfection. However, it is important to note that the detection results alone cannot conclusively differentiate between viable and non-viable bacterial cells when using nucleic acid methods [31]. Additional studies involving bacterial growth and enumeration would be necessary to provide a more definitive assessment [37].
Table 1 also highlights the presence of bacteria from the Pseudomonas genus, which have been associated with denitrification [38,39,40] and phosphate removal processes [39,41]. Additionally, Flavobacterium species belonging to the Bacteroidetes phylum were identified among the nutrient removal organisms. These species are known to be involved in denitrification, and comparative genomic analyses have revealed unique sets of denitrification genes in different Flavobacterium species, such as Flavobacterium columnare and Flavobacterium johnsoniae [42].
Furthermore, Table 1 reports the detection of ammonium-oxidizing species (Nitrosomonas marina and N. eutropha), sulfate-reducing bacteria (Desulfovibrio intestinalis and D. desulfricans) and phosphate-removing Staphylococcus aureus. These findings emphasize the presence of microorganisms involved in crucial nutrient removal processes within the wastewater treatment system.
Overall, the data presented in Table 1 underscores the impact of the activated sludge process on the microbial composition of the effluent and suggests the potential survival of nutrient removal organisms despite chlorine disinfection. However, further research is needed to differentiate between viable and non-viable cells and to gain a more comprehensive understanding of the microbial dynamics and their functional roles within the treatment facility.
Table
Table 1. Bacteria used for wastewater treatment detected at the final effluent from the treatment facility in this study.
Table 1. Bacteria used for wastewater treatment detected at the final effluent from the treatment facility in this study.
PhylumClassGeneraSpeciesRole in Wastewater Treatment ProcessReferences
ProteobacteriaBetaproteobacteriaComamonasC. testosteroniDenitrification[43,44]
NitrosomonasN. marinaNitrification[4]
N. eutrophaNitrification
Gammaproteobacteria AcinetobacterA. calcoaceticusPhosphate removal[45]
PseudomonasP. aeruginosaDenitrification[38,40,41,46]
P. stutzeriDenitrification
P. fluorescensPhosphate removal[39,45]
P. mendocinaPhosphate removal
P. Puti4daPhosphate removal
AeromonasA. hydrophilaPhosphate removal[47]
AlphaproteobacteriaRhodobacterR. capsulatusDenitrification[48]
ParacoccusP. denitrificansDenitrification[40,49]
HyphomicrobiumH. vulgareDenitrification[50]
H. methylovorumDenitrification
DeltaproteobacteriaDesulfovibrioD. vulgarisSulfate removal[51]
D. oxamicusSulfate removal
D. longusSulfate removal
BacteroidetesFlavobacteriiaFlavobacteriumF. columnareDenitrification[42]
F. johnsoniaeDenitrification
FirmicutesBacilliStaphylococcusS. aureusPhosphate removal[52]
Table 2 presents a comprehensive overview of disease-associated bacteria and pathogens detected in the effluent wastewater during this study. It is well-established that treated wastewater can contain numerous pathogenic bacteria, many of which are commonly transmitted through the fecal–oral route [53]. These organisms typically colonize the human intestine and are subsequently released into the environment via feces. While some of these bacteria are commensal and beneficial to their hosts, a significant proportion are enteric pathogens that pose concerns due to their ability to autonomously proliferate in the environment [53].
Numerous studies have identified diverse microbial groups in wastewater that have the potential to influence the microbial ecology of the surrounding environment [54]. Therefore, it is crucial to regularly perform microbiological assessments of effluent to identify potential pathogens. In this context, it is advisable to incorporate more sensitive technologies such as next-generation sequencing (NGS) alongside conventional coliform counts for a more comprehensive and accurate analysis.
By adopting these more advanced techniques, it becomes possible to gain deeper insights into the microbial composition of wastewater, detect a wider range of potential pathogens and better understand their dynamics within the treated effluent. This knowledge can significantly contribute to improved wastewater management strategies, ensuring the protection of public health and the environment.
Table 2 provides further insights by highlighting the detection of well-known bacterial species from genera associated with diseases [55,56]. These include Escherichia, Legionella, Pseudomonas, Mycobacterium, Clostridium, Aeromonas, Staphylococcus and Streptococcus. Notably, bacterial species from Vibrio, Leptospira, Salmonella, Campylobacter, Escherichia, Yersinia and Shigella are of significant concern due to their established association with disease development in the general population.
Additionally, bacterial species from Bacillus, Enterobacter, Klebsiella, Clostridium, Listeria, Pseudomonas, Staphylococcus and Streptococcus are considered of minor concern as they are primarily associated with opportunistic diseases in individuals with compromised immune systems [57,58].
By identifying and categorizing these bacterial species, the table sheds light on the potential risks associated with specific genera and species present in the examined microbial communities. These findings underscore the importance of maintaining effective wastewater treatment protocols to mitigate the spread of disease-causing bacteria and protect both the general population and immunocompromised individuals from potential health risks.

4. Conclusions

In order to enhance wastewater treatment processes, it is essential to develop a comprehensive understanding of microbial ecology. This study aims to contribute to this understanding by conducting a broad analysis of bacterial taxonomic diversity within the influent and effluent of an activated sludge sewage treatment plant in the North-West Province of South Africa. The utilization of DNA or RNA sequencing technologies, such as next-generation sequencing (NGS), holds immense potential for surveying the microbial quality of wastewater.
The findings of this study reveal that the effluent from the examined plant contains a diverse range of bacterial communities, primarily dominated by Proteobacteria, Actinobacteria and Firmicutes. These bacterial distributions may be influenced by their roles in organic matter removal and potential resistance to chlorination, among other factors. The identification of these phyla and classes aligns with previous reports on various activated sludge sewage treatment plants, supporting the consistency of these findings.
The presence of such broad bacterial taxonomic diversity in the final effluent suggests the potential survival of these organisms despite the disinfection protocols employed at the studied plant. It is important to acknowledge that the data presented in this study originate from a single wastewater treatment plant, and the limited number of samples represents a limitation of the study.
Further research is highly recommended to investigate microbial diversity comprehensively and assess the distribution of pathogens in wastewater treatment plants that discharge effluent into the environment for downstream users. This expanded research will provide valuable insights into microbial dynamics within wastewater treatment systems and their potential implications for environmental and public health.

Author Contributions

Conceptualization, S.M. and M.T.; methodology, S.M and M.T. validation, S.M., M.T., E.F.-K. and E.G.; formal analysis, S.M., V.M., M.T., E.F.-K. and E.G.; investigation, S.M. and V.M.; resources, S.M. and M.T.; writing—original draft preparation, S.M.; writing—review and editing, S.M., M.T., E.F.-K. and E.G.; supervision M.T., E.F.-K. and E.G.; project administration, S.M., M.T., E.F.-K. and E.G.; funding acquisition, E.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Research Foundation of South Africa, Grant number 112855 and the APC was funded by the Faculty of Natural and Agricultural Sciences, North West University, South Africa.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors would like to thank JB Marks Local Municipality, North-West, South Africa, for granting permission collect samples at the treatment facility.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Applmicrobiol 03 00053 g001 550
Figure 1. Charts showing relative taxonomic abundance of bacterial (a) phyla and (b) class, in influent and effluent samples based on sequencing data.
Figure 1. Charts showing relative taxonomic abundance of bacterial (a) phyla and (b) class, in influent and effluent samples based on sequencing data.
Applmicrobiol 03 00053 g001
Table
Table 2. Disease-associated bacteria detected at the final effluent of the treatment facility in this study.
Table 2. Disease-associated bacteria detected at the final effluent of the treatment facility in this study.
PhylumClassGenusSpecies
ProteobacteriaGammaproteobacteriaEscherichiaE. coli
E. fergusonii
SerratiaS. liquefaciens
S. odorifera
S. marcescens
Aeromonas A. hydrophila
A. media
A. jandaei
LegionellaL. pneumophila
L. jamestowniensis
L. erythra
Pseudomonas P. fluorescens
P. stutzeri
P. aeruginosa
ActinobacteriaActinobacteriaMycobacterium M. tuberculosis
M. leprae
FirmicutesClostridiaClostridiumC. septicum
C. butyricum
C. botulinum
BacilliStaphylococcusS. aureus
S. epidermidis
S. haemolyticus
StreptococcusS. salivarius
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Makuwa, S.; Green, E.; Fosso-Kankeu, E.; Moroaswi, V.; Tlou, M. A Snapshot of the Influent and Effluent Bacterial Populations in a Wastewater Treatment Plant in the North-West Province, South Africa. Appl. Microbiol. 2023, 3, 764-773. https://doi.org/10.3390/applmicrobiol3030053

AMA Style

Makuwa S, Green E, Fosso-Kankeu E, Moroaswi V, Tlou M. A Snapshot of the Influent and Effluent Bacterial Populations in a Wastewater Treatment Plant in the North-West Province, South Africa. Applied Microbiology. 2023; 3(3):764-773. https://doi.org/10.3390/applmicrobiol3030053

Chicago/Turabian Style

Makuwa, Stenly, Ezekiel Green, Elvis Fosso-Kankeu, Victor Moroaswi, and Matsobane Tlou. 2023. "A Snapshot of the Influent and Effluent Bacterial Populations in a Wastewater Treatment Plant in the North-West Province, South Africa" Applied Microbiology 3, no. 3: 764-773. https://doi.org/10.3390/applmicrobiol3030053

APA Style

Makuwa, S., Green, E., Fosso-Kankeu, E., Moroaswi, V., & Tlou, M. (2023). A Snapshot of the Influent and Effluent Bacterial Populations in a Wastewater Treatment Plant in the North-West Province, South Africa. Applied Microbiology, 3(3), 764-773. https://doi.org/10.3390/applmicrobiol3030053

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