Enhancing anaerobic digestion efficiency in dairy waste water treatment: a comprehensive review of enzyme-based pre-treatment by microorganisms in South Africa

Clean water refers to water devoid of any physical, chemical, or biological contaminants and can be consumed, used for domestic, and employed for cleaning purposes (Pandit & Kumar 2015). Access to clean water is considered a fundamental human right necessary for survival. Conversely, untreated water poses a heightened risk of illnesses like diarrhea, malaria, and other waterborne diseases. It is crucial to adhere to established discharge standards to ensure that all individuals have access to clean and safe drinking water (Hove et al. 2019). With the rapid population growth and industrialization, the dairy industry continues to grow due to the increasing population and urbanization influencing increased demands, resulting in more production of wastewater and the release of toxins into the environment. The dairy industry is a significant contributor to the agricultural sector on a global scale, and South Africa (SA) is certainly no exception. Addressing the pollution and challenges associated with this industry is critical considering continuous expansions over the few decades (Esterhuizen et al. 2012).

The substantial freshwater consumption in the dairy industry's cleaning, sanitation, and processing activities results in large volumes of wastewater, ranging from 0.2 to 10 L per litre of milk produced (Ates et al. 2017). The cleaning process involves several stages, such as cleansing the boilers, softening, and backwashing of filters which produces a specific type of wastewater known as cooling water. Additionally, detergents are employed in cleaning milk cans, tankers, and flooring, giving rise to a different kind of sewage called sanitary wastewater. Lastly, the water used in the cleaning equipment (CIP) process also contributes to industrial wastewater production (Preeti et al. 2017).

The toxins released in the wastewater are in the form of solids and liquids comprising different chemicals (Raghunath et al. 2016). As previously stated, these toxins are detrimental to the environment, threatening aquatic life and the flora surrounding the water sources and contributing to eutrophication. Due to this high volume of wastewater production and subsequent release of toxins, the dairy industry is considered one of the most polluting sectors among food industries (Borbón et al. 2014). Hence, the treatment of dairy waste water (DWW) is crucial in reducing overall damage to the environment.

Considering the breadth of recent publications and acknowledging the adverse effects associated with the disposal of huge volumes of untreated DWW, there is a need to choose the most efficient, minimal-footprint wastewater treatment options whilst adhering to municipal standards and environmental guidelines and fostering a sustainable economy. However, it is worth noting that existing treatment technologies often fall short of consistently achieving desired effluent standards and may entail significant costs. Nevertheless, these processes exhibit efficacy only within a limited scope of operations. Hence, fluctuations in flow rate and waste concentration impact the technological processes. Consequently, wastewater pre-treatment is essential before discharge to guarantee the effectiveness of subsequent stages in wastewater treatment facilities. This literature review paper assesses the performance and effectiveness of utilizing enzymatic pre-treatment using a bioremediation agent coupled with an anaerobic digester (A.D) for DWW treatment. The review paper also discusses the background of enzymatic pre-treatment, its associated challenges, and its integration into biological treatment processes. Also included are recommendations on DWW treatment and future research directions.

DWW contains high levels of organic pollutants, nutrients, and sediments that can cause environmental pollution if not treated appropriately. The high contaminants in DWW, if allowed to be released into freshwater sources without any treatment, destroy the water's features, making it less habitable and non-potable. There are many harmful consequences of these actions, such as the water body becoming a breeding area for various insects and ultimately giving birth to waterborne diseases such as malaria, yellow fever, dengue, and others (Mohebzadeh et al. 2013; Al-Wasify et al. 2018; Joshiba et al. 2019). According to Shams et al. (2018), the disposal of casein from dairy plants is a major culprit in freshwater contamination, leading to foul-smelling, black-colored sludge that harms aquatic ecosystems. The excessive nutrient content triggers accelerated growth of algae and bacteria, depleting oxygen levels in the water and consequently causing a decline in aquatic life (Bhuvanendran et al. 2022).

Overall, selecting an appropriate treatment technology hinges on understanding the specific characteristics of the DWW, the level of contaminants, and the prevailing conditions for optimal mitigation. Table 1 highlights the main issues with DWW and the need to treat it to avoid environmental harm.

DWW, known for its high-fat content, contains a significant amount of FOGs. DWW treatment encounters challenges like poor sludge settleability, nitrogen, and phosphorous removal complexity and substantial scum production (Andrade et al. 2013). Upon release into sewage systems, the presence of abundant FOGs, alongside solids such as cheese remnants, coagulated milk, curd fragments, and equipment cleaning residues like sand or soil, can engender blockages, potentially leading to pipeline bursts (Otsuka et al. 2020). Thus, removing these contaminants of high-fat content wastewater is imperative before subjecting it to any sustainable biological treatment approaches. By employing enzymes, it is possible to reduce the FOG contents of the raw DWW influent. This reduction has a cascading effect, leading to a decrease in the organic content. Hence, the biological treatment process can operate without a significant energy requirement, reducing the overall treatment time. According to Bella & Rao (2023), pre-treatment reduces the FOG content, increases solubilization, improves the digestion rate, and decreases the raw influent's organic content. Biological treatment of high-fat content wastewater, such as using an AD, is effective in the reduction of substrates and organic content. However, the FOGs pose numerous challenges, as mentioned above, thus requiring assistance from a pre-treatment stage to enhance the treatment of the raw influent (Mobarak-Qamsari et al. 2012; Harris & McCabe 2015).

Pre-treatment plays a key role in enhancing the effectiveness of conventional wastewater treatment processes. Several studies have examined the different methods utilized in pre-treatment procedures, thereby supporting biodiversity and ecosystems. Certain techniques within this process demand substantial energy consumption, increased air circulation, and are time intensive. However, to mitigate these challenges, integrating biological pre-treatment processes anchored to reactors has proven effective and efficient for treating DWW (Joshiba et al. 2019). An example often cited as representative of such a reactor is the Up-flow Anaerobic Sludge Blanket (UASB), which has been extensively documented. The UASB reactor stands out as a highly efficient and cost-effective solution for wastewater treatment. It has demonstrated remarkable efficacy in treating concentrated wastewater with high organic loads. Kaviyarasan (2014) describes the UASB as a system containing microbial granules, small agglomerations of microorganisms (0.5 to 2 mm in diameter), formed within the sludge blanket of the UASB. Due to their weight, these granules resist being washed out by the flow. Anaerobic digestion (AD) facilitated by bacteria in the sludge converts organic matter into biogas. The natural mixing of the sludge occurs through rising bubbles, eliminating the need for machinery while sloping walls push down material reaching the top of the tank. Wastewater flows into the UASB reactor from the bottom and flows upward. UASB reactors are particularly appealing because they can treat wastewater with broader strengths and higher suspended solids.

The advantages of using a UASB are that it is cost-effective and requires little space as it can be constructed underground, UASB has shown that it can reach high removal rates in high-strength wastewater, the biogas that is produced can be used as a source of energy, it produces small amounts of sludge, there are fewer CO₂ emissions from the system thus air pollution is reduced, there is no need for temperature control as heat is released during the methanogenesis stage and the effluent produced is high in nutrients thus it can be used for agricultural purposes (Kaviyarasan 2014; Goli et al. 2019).

However, drawbacks highlighted by Goli et al. (2019) include a long startup period, substantial seed sludge requirement, dependence on skilled operators, partial pathogen removal, and hydrogen sulfide emission causing foul odors (Sinha et al. 2019). Accumulation of FOGs in the reactor can obstruct gas movement, affecting performance. A modified UASB reactor proposed by Bhuvaneshwari et al. (2022), equipped with a scum extraction device and a lamella settler, effectively reduces biomass washout, and eliminates FOGs, preventing blockages. Most UASB systems operate at mesophilic (25–45 °C) or thermophilic conditions (45–65 °C), but maintaining such temperatures requires significant energy, rendering it expensive and often impractical. Organic debris buildup during no wastewater inflow compromises system efficiency, emphasizing the need to reduce debris during downtime and intermittently run the system to enhance biological conversion efficiency. Combining the UASB system with another treatment, as Ji et al. (2020) demonstrated with an anaerobic baffled reactor (ABR), can achieve high COD removal rates.

When there is no inflow of wastewater, organic debris tends to build up within the sludge bed, which can cause the system to not operate at its optimum, thus reducing the organic debris in the sludge bed when there is no flow to increase the system's efficiency. Intermittently running the system also improves biological conversion efficiency. Another way of increasing the system's efficiency is to combine it with another treatment as well. Ji et al. (2020) reported that a combined system of an ABR and a UASB reactor achieved a COD removal rate of 98% (Figure 4).

The pre-treatment stage involves a series of physical, chemical, and biological processes designed to remove or neutralize specific contaminants from wastewater (Goli et al. 2019). Physico-chemical pre-treatment has been researched and reported to be effective and fast. However, the drawbacks outweigh the former: the reagents needed are costly and hazardous, the reaction conditions are severe, and it requires a large amount of energy (Arvanitoyannis & Giakoundis 2006; Crini & Lichtfouse 2019; Musa & Idrus 2021). Considering these challenges, the biological route for pre-treatment is a better option; this includes using enzymes that comply with environmental regulations and green economy goals (Cammarota & Freire 2006; Mobarak-Qamsari et al. 2012; Adulkar & Rathod 2015). A study investigating the pre-treatment of DWW with lipase Z coupled with the ultrasound irradiation technique by Adulkar & Rathod (2014), reported that coupling the enzymatic pre-treatment with ultrasound increased the rate of hydrolysis and lowered the reaction time. The optimum conditions used in this study were a 0.2% (w/v) enzyme load, a temperature of 30 °C, an ultrasonic power of 165 W, a frequency of 25 kHz, and a mixing speed of 200 rpm.

Hernández et al. (2015) reported that the combination of acid and enzymatic pre-treatment effectively broke down microalgal cell walls and converted carbohydrates into monosaccharides for bioethanol production. Acid hydrolysis is particularly effective at disrupting cell walls, increasing enzyme efficiency. The authors reported that the best results were achieved through a combination of H₂SO₄ with enzymatic hydrolysis, with the highest sugar release (S.R.) in C. sorokiniana (128 mg/g D.W.) and N. gaditana (129 mg/g D.W.). S. almeriensis had the highest S.R. from acid hydrolysis with H₂SO₄ for 60 min (88 mg/g D.W.). Enzymatic hydrolysis with amylases of C. sorokiniana previously suspended in 0% sulfuric acid released a significant concentration of monosaccharides (101 mg/g DW).

Gomes et al. (2011) reported that lipase pre-treatment increased the organic load of high-fat wastewater in a hybrid UASB reactor. Mendes et al. (2006) found that using pancreatic lipase drastically reduced the size of fat particles in pork increased LCFAs (long-chain fatty acids) in the liquid phase and decreased the digestion time of slaughterhouse wastewater. Results of pancreatic lipase pre-treating high-fat wastewater showed that the fat particles were reduced by 60%, and a 4-h pre-treatment increased the free LCFAs concentration to a maximum of 15.5 mg L⁻¹.

Adulkar & Rathod (2015) reported that enzymatic pre-treatment using lipase Z of synthetic DWW before AD resulted in a reaction conversion of 75% and COD removal of 72% under optimum conditions. These conditions were reported to be 0.2% (w/v) enzyme load and a temperature of 30 °C. Sodium chloride (NaCl) was used as an emulsifying agent to promote the enzyme activity, increasing the reaction rate by 30%.

A study using lipase from Aspergillus niger in the pre-treatment of FOG-rich food waste found that biomethane production increased and the volatile solids decreased. Biomethane production was reported to be 0 mL g⁻¹ at 0.5% (w/w) lipase concentration, and the digestion time was reduced by 10–40 days (Meng et al. 2015, 2017). Salama et al. (2019) stated that pre-treatment of FOG with the applicable lipase can increase the rate of hydrolysis and the production of biomethane.

Based on the case studies, it is evident that employing an enzymatic pre-treatment method for high-fat content wastewater yields multiple advantages and effectively manages the wastewater. In the biological pre-treatment approach, the energy expenditure is reduced, yet the use of enzymes leads to an increase in the overall operational cost (OPC). Considering the OPC, the expenses for chemicals and enzymes are significant, resulting in a higher combined biological and chemical OPC. Consequently, evaluating its energy consumption which directly affects the OPC is important. From Figure 5, it can be seen that the energy cost implications for chemical and physical pre-treatment is medium cost whereas enzymatic pre-treatment is of low energy consumption according to Preethi et al. (2023). This indicates that enzymatic pre-treatment is the best value for money amongst these pre-treatments. Table 3 provides further instances for reference. This enzymatic pre-treatment substantially mitigates the primary challenge associated with high-fat content wastewater, namely FOGs, thereby facilitating the smoother operation of the overall treatment process (Feng et al. 2021). Moreover, the byproduct generated during this treatment proves advantageous and boosts the production of biomethane, which can be utilized as an energy source.

Biological pre-treatments, also known as pre-hydrolysis and two-stage digestion, involve the modification or degradation of organic materials by using bacteria, fungi, or enzymes. The use of biological pre-treatment methods can have several advantages, including reduced energy consumption, milder operating conditions, and the possibility of generating value-added products from waste materials. The effectiveness of these methods depends on multiple factors, such as the composition of the material, the specific microorganisms or enzymes used, and the desired final product.

Enzymatic pre-treatment represents a potent and efficient solution for handling the complexities of DWW. Enzymes are biological catalysts that play an essential role in biological pre-treatment by catalyzing the breakdown of biomass components, thereby increasing the reaction rates. By utilizing the catalytic abilities of enzymes, specific organic compounds present in the wastewater can be efficiently broken down, resulting in improved biodegradability and a reduced environmental impact. Moreover, enzymatic pre-treatment can significantly enhance the performance of other treatment processes, such as biological treatment and membrane filtration, by breaking down complex organic compounds that would otherwise be difficult to degrade (Ahmad et al. 2019).

This section focuses on discussing enzymatic pre-treatment tailored for DWW. It will delve into the enzymes employed, their modes of action, and the parameters governing their activity in DWW. Additionally, the section will explore the myriad of benefits and challenges associated with implementing enzymatic pre-treatment in DWW.

Once the enzyme–substrate molecule is formed, the enzyme catalyzes the conversion of the large substrate into smaller molecules. The enzyme accelerates this reaction by weakening and breaking down the chemical bonds within the sizeable organic molecule (Bella & Rao 2023). These processes have high reaction kinetics and reduce the time required for the substrates to travel into cells, making the process more effective (Feng et al. 2021).

Ligand binding is the interaction between the ligand (molecule/ion) and the receptor but within in enzymes where the active sites are located deep within the protein structure an extra step is required. This is because potential substrates need to traverse through the protein's interior to reach the active site. In contrast to enzymes with active sites exposed on the protein surface, this structural arrangement offers more interaction opportunities between proteins and ligands. This is because the substrate must navigate through a series of tunnels before binding to the active site. The core idea of this model is that in enzymes with concealed active sites, the ligand binding process involves two distinct steps. First, the ligand must migrate through the protein's body, and then it can bind to the active site. By breaking down the process in this manner, the theory suggests that, in addition to compatibility between the ligand and the active site, there must also be compatibility between the ligand and the binding tunnel (Kingsley & Lill 2015).

Lipase is an enzyme that specifically targets the fats, oils, and grease molecules in wastewater, making it a vital enzyme in the treatment of high-fat content wastewaters such as dairy (Mobarak-Qamsari et al. 2012; Feng et al. 2021). It catalyzes the hydrolysis process of carboxyl ester bonds present in triacylglycerol into free long-chain fatty acids and glycerol. The process of lipase follows the ping-pong bi–bi mechanism (Figure 7). The initial step in this mechanism is the combination of the enzyme with an acyl donor, which is a triglyceride in this scenario which then forms a lipase–triglyceride complex. The isomerization process converts the lipase-triglyceride complex into an intermediate complex and produces glycerol. The binary complex is formed when the intermediate complex combines with three molecules of water. In the last step, fatty acids are produced by unimolecular isomerization, and then the enzyme is regenerated, and the process continues (Liew et al. 2020).

Amylase is introduced into the wastewater to break down carbohydrates, also called polysaccharides; they are made up of large chains of simple sugars. Amylase breaks these large chains of simple sugars into small polymers comprising glucose (Liew et al. 2020; Osho et al. 2021).

Protease is an enzyme that breaks down the proteins within the wastewater. Proteins are polymers which comprise amino acids joined by peptide bonds. This enzyme hydrolyses these peptide bonds to break the protein down into simpler compounds such as polypeptides and amino acids. The protein in wastewater constitutes high amounts of total organic carbon and organic matter. Thus, a breakdown of proteins within the wastewater can reduce the total organic carbon (TOC) of the wastewater as well as produce simple sugars which can be used in the methanogenesis process in the AD (Facchin et al. 2013; Pandey et al. 2017; Liew et al. 2020).

Conclusively, these enzymes are pivotal in the treatment of DWW. Their enzymatic properties show great promise in effectively managing high-fat content wastewater, demonstrating their ability to degrade fats and greases and enhance anaerobic biodegradability (Mendes et al. 2010; Konkit & Kim 2016). Consequently, they facilitate smoother and more efficient treatment processes, contributing to developing environmentally friendly and highly effective methods for treating DWW.

Temperature is a crucial factor in the performance of enzymatic reactions as it is directly related to the solubility, the fluidity of the reactive media, and the viscosity of the substrates, which all affect the mobility of reactants (Baena et al. 2022). The optimum temperature range is between 37 and 40 °C; if the temperature increases, then the enzyme activity slowly depletes until there is no activity and can lead to thermal denaturation as the internal bonds of the enzyme are sensitive to high temperatures (Liew et al. 2020; Baena et al. 2022). The enzyme will be inactive if the temperature is below the optimum temperature. Skliar et al. (2019) reported that the optimum temperature conditions of lipase Rhizopus japonicus depended on the correlation between (i) the influence of the temperature on the speed of reaction and (ii) the effect on enzyme denaturation rate.

When using enzymes for wastewater treatment, it is crucial to emphasize that increasing the enzyme dosage can speed up reactions by enhancing the number of available active sites. According to Mendes et al. (2010), an increase in lipase concentration led to a rise in free fatty acid levels. Yang et al. (2010) found that increasing the enzyme dosage from 3 to 6% (w/w) enhanced the reduction in Volatile Suspended Solids (VSS). However, the authors also observed that any dosage exceeding 6% (w/w) did not yield further reductions in VSS. Liew et al. (2020) suggested that the optimal enzyme dosage ranges between 1 and 2% (w/w).

In wastewater treatment, the substrate concentration plays a crucial role in the interaction between the substrate and the enzyme. As the substrate concentration increases, there are more frequent collisions between the substrate and the enzyme. This leads to a faster interaction between the substrate and the enzyme up to a certain point. However, further increases in substrate concentration do not bring any additional benefit beyond this point (Robinson 2015). This is because the enzyme's active site becomes fully saturated with the substrate. As a result, when a new substrate tries to attach itself to the enzyme, it has to wait for the enzyme–substrate complex to release the product, thereby freeing the enzyme for further interaction.

The electron acceptor and donor molecules can significantly affect enzyme activity by influencing the redox (reduction–oxidation) reactions that enzymes facilitate. Electron acceptors are chemicals such as sulfates, nitrates, and high-valence metallic ions within the biochemical processes. The electron donor is usually comprised of organic matter, low valence metals, sulfides, and ammonia (Li et al. 2019; Tian & Yu 2020; Ying et al. 2021).

The enzyme will increase the hydrolysis rate, leading to an increase in readily biodegradable substrates. Breaking the complex substrates into smaller substrates allows for an increase in electron donors. Thus, with the increase in the electron donors, the biomass will increase linearly.

Establishing the ideal pH is crucial in enzymatic pre-treatment for DWW treatment. It is imperative to ascertain the optimal pH level, as the enzyme's functionality is compromised if the pH deviates from the optimal range of 7 to 8. According to Liew et al. (2020), when lipase is used at the optimum pH, it performs at its maximum and shows negative potential in the active site, which causes the ionized fatty acids to repel one another, which results in a fast release of lipolysis reaction products from the interface. This has a positive effect on the pre-treatment process because when the enzyme is at its optimum conditions there is an increase in the lipolysis reaction which helps in the breakdown of fats which is crucial in the removal of fats in the pre-treatment stage of treatment of high-fat content wastewaters.

Generally, and in the context of enzymes' role in DWW treatment, enzyme inhibition is distinct from denaturation as it involves the interference of a reagent with the active site region, leading to a specific action. This process is classified into two main types: (i) reversible inhibitors and (ii) irreversible inhibitors. The former, in turn, can be further categorized into distinct categories, including competitive, non-competitive, and uncompetitive inhibitors. In competitive inhibitors, the inhibitor mirrors a substrate and can bind to the same active site as the substrate, thus creating competition for the binding. Competitive inhibitors can be displaced from the active site if there is a high substrate concentration, thus restoring the enzyme activity. Non-competitive inhibitors bind with the enzyme but at a different site away from the active site. It does not block the substrate from binding with the enzyme at the active site; it, however, reduces the catalytic ability of the enzyme, subsequently reducing the enzyme. Uncompetitive inhibition is rare and occurs after binding the enzyme and substrate. When an inhibitor forms a permanent bond with an enzyme, it is categorized as an irreversible inhibitor. Consequently, irreversible inhibitors possess high toxicity levels, an example of this inhibitor is organophosphorus compounds such as diisopropyl fluorophosphate (Haggag et al. 2013; Robinson 2015; Bhatia 2018).

Enzymes play a pivotal role in the initial treatment of diary wastewater, significantly aiding in the reduction of FOGs, which are recognized as essential substrates for fostering biomethane generation. Salama et al. (2019) reported that the high content of lipids within FOGs causes a higher yield of biogas due to the higher convertibility of 94.8% to biogas compared to 50.4% due to carbohydrates and 71% due to proteins. Enzymatic pre-treatment also reduces the organic matter within the wastewater, thus reducing the organic load going into an A.D (Bella & Rao 2023). A study by Samarasiri et al. (2019) demonstrated that the utilization of enzymes effectively reduces the energy input necessary to initiate a reaction. This not only accelerates the reaction rate but also enhances the hydrolysis process. The researchers also re-affirmed that pre-treatment with lipase decreased organic content within DWW effluent and facilitated biogas generation. Enzymes offer advantages over microbes in pre-treatment organic-rich wastes as they do not require an acclimatization phase to the biomass, leading to a faster process. Additionally, enzymes are biodegradable proteins that break down independently without generating new biomass. Enzymes can operate under mild conditions (i.e. low temperature and a neutral pH) and are effective in various environmental conditions like pH and temperature (Liew et al. 2020). Enzymatic pre-treatment stands out as a more environmentally sustainable and economically viable approach for wastewater treatment, particularly when juxtaposed with other methods reliant on chemical agents.

One significant drawback of utilizing enzymatic pre-treatment is the associated cost implications. As the usage of enzymes increases, there is a corresponding increase in the required purchase of enzymes. Thus, determining an optimal dosage becomes essential. According to Liew et al. (2020), while enzymes demonstrate functionality across various environments, such as different pH and temperatures, they are prone to denaturation and inactivity beyond their designated operational parameters. Consequently, careful monitoring and maintenance of the ideal conditions are consistently necessary. The enzymatic process's speed can be relatively slow, contingent upon the type of wastewater or the specific targeted substrate, necessitating frequent replenishment (Harris & McCabe 2015; Rodriguez et al. 2015; Neumann et al. 2016). Baena et al. (2022) have identified a limiting factor, indicating a lack of compatibility between reactants and the bio-derived catalyst. To address this, using surfactants in the reactive media to achieve compatibility is recommended, albeit with some associated drawbacks.

Various studies have investigated the efficacy of different treatment methods for DWW. Among these, aerobic treatment approaches have been emphasized in research conducted by Scott & Smith (1997); Carta-Escobar et al. (2004); Kolev Slavov (2017); Joshiba et al. (2019). However, when considering DWW treatment, AD emerges as a more favorable option than aerobic digestion. This preference stems from its ability to yield biogas as a byproduct, its reduced spatial requirements during construction lowered aeration demands resulting in decreased energy consumption, and the added advantage of reduced sludge and biomass generation, as highlighted by Adulkar & Rathod (2014). Notably, within the dairy sector, Carvalho et al. (2013) assert that anaerobic processes are particularly well suited for addressing wastewater with high organic loads. Supporting this notion, Kolev Slavov (2017) presents findings indicating that anaerobic bacteria outperform aerobic bacteria in DWW treatment. Aerobic processes are compromised due to rapid acidification and filamentous growth attributed to elevated lactose levels and limited water buffer capacity, as discussed by Ahmad et al. (2019).

Leal et al. (2006) reported that when there is an increase in FOGs in the wastewater, the COD removal rate decreases in the UASB, but when the wastewater is pre-treated with an enzyme, the COD removal rate is above 90%. A study by Bhange & Suke (2018) investigated the effect of lipase of different dosages on high-fat DWW. They reported that there was an average removal rate of 97% of FOG, 67% of COD and 38% of T.S. According to Cammarota et al. (2001), when DWW with high levels of FOGs is treated in a UASB reactor, the effluent is high in turbidity, VSS and achieves a COD removal rate of <50%. The authors reported that pre-treated DWW with Penicillium-restricted lipase showed a higher % COD removal rate of 90% when treated with the same UASB reactor. AD treatment is prompt to release high amounts of nutrients which can be recovered using recovery technology such as struvite formation, which acts as a slow-release fertilizer. In some areas of SA, the struvite and stabilized sludge are used for the growth of animal fodder (Nqayi et al. 2023), which is useful in the context of resource recovery from dairy waste.

According to existing literature, lipase stands out as the commonly employed enzyme in treating DWW due to its ability to breakdown high-fat content (Ahmad et al. 2019). This enzyme effectively acts as a pre-treatment agent for DWW, augmenting the hydrolysis of FOGs within the wastewater before its introduction into the A.D. The remaining FOGs further promote the production of biomethane. The performance analysis of lipase is outlined in Table 3, revealing its favorable impact on DWW and its assistance in the AD treatment process. Bella & Rao (2023) and numerous other researchers emphasize the necessity of pre-treatment techniques for complex dairy substrates, particularly to expedite the hydrolysis stage. Gutu et al. (2021) confirm that integrating an enzymatic pre-treatment step before A.D enhances the reactor's efficiency, amplifies biogas production, and accelerates the breakdown of FOGs. Nonetheless, a key limitation associated with using enzymes is their cost implications, particularly when dealing with large volumes of DWW, necessitating a higher dosage and consequent continuous procurement of enzymes. Consequently, various scholars propose exploring new, more economical enzyme options to ensure the sustainability of enzymatic pre-treatment methods.

Genetically modified microbes (GMMs) find exclusive application in South Africa's agricultural industry, primarily driven by the urgent need to address challenges such as the burgeoning population, food scarcity, malnutrition, and soaring production costs. The GMMs are mainly used to enhance the cultivation of staple crops such as maize, soybeans, and cotton. Notably, the use of GMMs yields substantial benefits, an upsurge in the agricultural sector's revenue, thereby making a significant contribution to South Africa's overall economy. These modified microbes boost crop yields, minimize crop damage, and enhance food quality. Moreover, they contribute to reducing carbon dioxide emissions by curbing the requirement for insecticides and herbicides. Additionally, the adoption of genetically modified technology has led to a considerable reduction in fuel consumption on farms (Bothma et al. 2010; Muzhinji & Ntuli 2020; Rozas et al. 2022).

Exploring new, affordable enzymes is crucial for improving the overall effectiveness of pre-treating wastewater. For instance, Eco-Flush and Morma are prime examples that exhibit potential in the reduction of FOGs in wastewater containing high-fat content.

Eco-Flush, a South African product, serves the purpose of hydrolyzing FOGs in kitchen sinks and drains. This biological treatment is facilitated by a specialized group of bacteria, ensuring a swift, efficient, and eco-friendly breakdown of the FOGs. The product's development revolves around a completely natural consortium of bacteria (Ergofito 2019). The enzyme Eco-Flush is composed of microorganisms that have been cultivated and preserved in a dormant physiological state. The microorganism has a complex structure comprising various bacteria, such as anaerobic, aerobic, sulfur, and nitrifying oxidized bacteria, which are combined with enzymes, fungi, and water (Dlamini et al. 2021; Dyosile et al. 2021). The natural constituents of the product come from amino acids and glucides, which stimulate the microorganisms to produce enzymes required to break down the FOGs of the wastewater oxidize NH₃ into and alongside the elimination of odor-producing organisms. Upon the introduction of Eco-Flush into the wastewater, the microorganisms initiate a reaction, instigating the production of enzymes that commence the bio-delipidation process. This process involves the disruption of bonds between triglycerides and phospholipase, thereby separating the fatty acids from the wastewater (Dyosile et al. 2021).

On the other hand, Morma, a U.S.-based company, has developed a range of products, including a liquid bacteria digester designed to assist in treating effluents. This liquid bacteria digester is a combination of five different live spore-forming bacteria. This bacterium, when added to different sources of waste, will produce various enzymes, which include protease (protein), amylase (starch & carbohydrates), lipase (fat & grease), esterase (fat), cellulase (cellulose, wood, paper), xylanase (plant material), and urease (urea). Although it produces all these enzymes, the bacteria will only produce as many as needed to complete the waste source's digestion. This bacterium works best at a pH range of 5–9 and a temperature range of 12–35 °C. The advantages of employing this bacterium include its rapid activation within an hour of introduction to the waste source.

Furthermore, the aerobic and facultative bacteria within this liquid formula can function effectively in the presence of either oxygen or nitrates. Compared to other naturally occurring bacteria, it exhibits greater resistance to fluctuations in temperature and pH. Additionally, its non-toxic nature eliminates any harmful acids, and it offers a pleasant fragrance that counteracts the foul odor of the waste source.

In the realm of wastewater treatment, Eco-Flush has been extensively studied, yielding significant results in the treatment of poultry slaughterhouse wastewater (Bingo et al. 2021; Dlamini et al. 2021; Dyosile et al. 2021; Gutu et al. 2021; Mdladla et al. 2021; Meyo et al. 2021). However, Morma, despite its capabilities, has not yet been utilized in any wastewater treatment procedures.

In conclusion, implementing innovative solutions such as Eco-Flush and Morma shows promise in boosting wastewater pre-treatment efficiency. Eco-Flush, a natural South African product, employs a diverse consortium of microorganisms to rapidly and ecologically degrade fatty, oily, and odorous compounds in wastewater. Its enzymatic approach effectively breaks down FOGs whilst complimenting other organisms that are common in high-strength wastewaters and converts ammonia (NH₃) into less harmful byproducts such as and (Meyo et al. 2021). On the other hand, Morma, a U.S. product, utilizes a versatile liquid bacteria digester that can adapt to different waste sources. This bacterium efficiently digests various waste components, adjusting its enzymatic activity based on the waste's composition. Its adaptability, quick action, and ability to thrive in diverse environmental conditions make it a compelling option for wastewater treatment. Overall, introducing these innovative solutions represents progress in wastewater management, promising improved purification results and a more sustainable approach to addressing contaminants and odor concerns.

Although anaerobic treatment is typically preferred over aerobic treatment in managing DWW, it can still be employed as a preliminary step before AD. This preliminary aerobic treatment can accelerate hydrolytic activities during the initial stage, improving hydrolysis and increasing methane production. The use of aerobic treatment prior to a two-stage AD process showcased that in the second stage, the methane production was much higher than without aeration (Rafieenia et al. 2017). Furthermore, it can also be employed as a post-treatment measure following AD, aiding in the enhanced removal of nutrients. Some future recommendations for the potential advancement in the treatment of DWW as well as implementations in SA are highlighted below.

1. Enzymatic pre-treatment has proven to be highly effective in the removal of solids before the implementation of biological treatment, such as AD. The use of enzymes catalyzes the hydrolysis process, demonstrating high substrate and reaction specificity. This specificity results in minimal side reactions and the generation of negligible or no waste byproducts (Liu & Smith 2021). Coupling biological treatment with enzymatic pre-treatment holds the potential for the development of more efficient wastewater treatment systems. Modern technologies can play a crucial role in improving the physiological conditions, including temperature and pH, ensuring that the enzymes operate optimally. Moreover, these advancements can reduce costs and decrease energy consumption (Pandey et al. 2017). Feng et al. (2021) suggest conducting further studies using various enzymes for treating different sources of natural wastewater rather than synthetic wastewater. Considering the potential cost constraints associated with treating large volumes of wastewater, the authors propose exploring new enzymes that are both efficient and economically viable.

2. Eco-Flush has found application in the treatment of PSW (Poultry Slaughterhouse Wastewater), where it serves as a pre-treatment method. Gutu et al. (2021) reported that Eco-Flush degrades the FOGs present in the PSW before undergoing treatment in the AD. Meyo et al. (2021) observed that using Eco-flush during the pre-treatment stage reduces the TSS and COD of raw PSW. Subsequent treatment using the expanded granular sludge bed reactor (EGSBR) led to an over 80% removal of both FOGs and TSS, although the average removal of COD was only 60%. As PSW is characterized by a high-fat content, exploring the application of Eco-Flush in DWW could significantly mitigate the high-fat content before the implementation of biological treatment. The rapid activation rate of Eco-flush enables the removal of FOGs within 24 h, depending on the volume of wastewater being treated. Despite showing promising results, further optimization remains necessary and warrants continued investigation.

3. While Morma has traditionally been utilized as a drain cleaner in industrial settings to break down fat, dirt, and other substances, its potential application in wastewater treatment has yet to be explored. Morma contains hydrolytic enzymes that can potentially reduce FOGs in wastewater, making it a promising addition for treating high-fat content wastewater.

4. In South Africa, GMMs have primarily been confined to the agricultural sector, exhibiting significant potential in enhancing crop yield and fostering industry growth. However, the adoption of GMMs for wastewater treatment has only gained momentum in the last decade, primarily through studies utilizing Eco-Flush for treating PSW. Given their cost-effectiveness and eco-friendly nature, further in-depth exploration of GMMs in wastewater treatment is imperative. Investigating the application of Morma in the pre-treatment of DWW for FOG reduction, coupled with subsequent biological treatment, warrants attention. Employing design software can aid in determining the optimal conditions, including dosage, time, and temperature, for utilizing Morma during the pre-treatment stage.

The dairy industry plays a significant role in global agriculture, but its processing activities generate substantial wastewater. Unfortunately, the high levels of contaminants in DWW, including BOD, COD, TSS, TN, TP, and FOGs, make it one of the most polluting sectors in the food industry. These contaminants pose a significant threat to the environment and aquatic life and contribute to eutrophication, characterized by an overabundance of nutrients and oxygen depletion in freshwater sources. The substantial amount of FOGs in DWW presents safe disposal and treatment challenges. However, FOGs exhibit high convertibility to biogas (94.8%), making them a valuable resource for biogas production compared to carbohydrates (50.4%) and proteins (71%). Studies on DWW treatment consistently favor AD over aerobic digestion due to its ability to yield biogas, reduced spatial requirements, lower energy consumption, and decreased sludge and biomass generation. Despite these advantages, the high-fat content in DWW can lead to blockages in the reactor, increasing treatment time and energy demand. Therefore, a pre-treatment stage is essential to reduce contaminants before entering the AD. Enzymatic pre-treatment emerges as an efficient solution, as enzymes catalyze the breakdown of organic compounds, effectively reducing FOG content, increasing biogas production, improving digestion rates, decreasing organic content, and enhancing anaerobic biodegradability. However, EMBs faces influencing factors and mechanisms, including the enzyme–substrate complex, temperature, enzyme concentration, substrate concentration, electron donor/acceptor availability, pH, and inhibitors. Commonly used enzymes in DWW pre-treatment are lipase, amylase, and protease. Lipase, particularly, proves effective in hydrolyzing FOGs before introducing the wastewater to A.Ds, leading to enhanced performance and biogas production. A limitation of enzymatic pre-treatment is its cost, which can be significant for large volumes of wastewater. This necessitates further research into more economical enzyme options. Exploring affordable enzymes such as Eco-Flush and Morma, which exhibit potential in reducing FOGs in high-fat content wastewater, represents a way forward in advancing enzymatic pre-treatment of DWW. In conclusion, this review underscores the imperative for ongoing collaboration among researchers, industry experts, and policymakers to promote the adoption of enzyme-based pre-treatment as a transformative and sustainable approach to managing DWW in South Africa and beyond.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.