Biodegradation of dairy wastes using crude enzymatic extract of Yarrowia lipolytica ATCC 9773

Effluents generated by the food industry have become a serious environmental concern. Bioremediation is a biological process developed as an alternative for the treatment of contaminated areas. In current research, the biodegradation of fat, Biochemical Oxygen Demand (BOD5), Chemical Oxygen Demand (COD) and total solids were evaluated in dairy waste employing enzymatic extract of Yarrowia lipolytica ATCC 9773 as biological agents. All the variables were determined following the specifications of the Standard Methods of the American Water Works Association. Enzymatic extract of Y. lipolytica at different concentrations (8, 12 and 16.0%) was used in a fermentative medium at two pHs (5.0 and 6.5) for 32 h. The highest percentages (%) of fat (82.88), BOD (43.32), COD (44.3) and total solids (13.58) removal were obtained using an inoculum concentration of 16% at pH 5.0 for 32 h of fermentation. These results may have industrial relevance for the reduction of contamination of industrial effluents with high levels of fat and other contaminants.


INTRODUCTION
The food industry is a sector with a high incidence of environmental pollution. Different industries such as dairy, meat and vegetable oil refining produce large amounts of wastewater. The high oil content of these increase the spoilage of some ecosystems (Porwal et al., 2015).
Dairy wastes are pollutants that affect the environment when they are discarded without adequate treatment (Liu et al., 2015b). In the last decades, fatty effluents have been released to the environment without previous treatment (Kumari et al., 2017;Tarón-Dunoyer et al., 2017), which affects public health and environmental sustainability. Considering the aforementioned, the treatment of fatty effluents is an economic and hygienic requirement.
Many of these effluents require pretreatment in order to remove incompatible substances before they are discharged into sewer systems (Kumari et al., 2017;Pilusa et al., 2013). The main components present in wastewater are: oils, fats and long-chain fatty acids, which represent a great problem in the pretreatment due to the fact that they are contaminants of aquatic ecosystems (Becerra-Gutiérrez et al., 2015;Fachin et al., 2013;Abass et al., 2011) Currently, there has been an emphasis on finding new biotechnological alternatives for the treatment of wastewater that also minimize the adverse effects previously mentioned. Biological treatment is one alternative used to decontaminate wastewater, where lipolytic properties of living organisms are employed to eliminate high-fat waste from the aquatic ecosystem (González et al., 2012;Kushwaha et al., 2011). Various microorganisms such as filamentous fungi, bacteria and yeast are well known as lipolytic microorganisms. Within this group of microorganisms, the yeast Yarrowia lipolytica is highlighted due to its extracellular and intracellular activity (Darvishi et al., 2017). Its lipase production depends on the medium composition and environmental conditions (Deive et al., 2010).
Yarrowia lipolytica is an adequate biological agent for biodegradation of substrates with a high fat content (Lopes et al., 2018). Y. lipolytica has been approved as GRAS (Generally Recognized As Safe) in several industrial processes. Yarrowia sp. have been isolated from lipidrich foods such as cheese and olive oil as well as from wastewater (Aloulou et al., 2007). The preference of Y. lipolytica for these substrates has been attributed to the efficient production and secretion of proteolytic enzymes (Fickers et al., 2005;Liu et al., 2015a;Brigida et al., 2014). In this sense, there are no references in the literature regarding the use of crude enzymatic extract for the biodegradation of dairy waste. Hence, this research focused on the utilization and evaluation of the biodegradation capacity of the crude enzymatic extract of Yarrowia lipolytica ATCC 9773 in dairy waste.

Industrial dairy waste
The dairy waste sample was obtained from a dairy industry located in Valledupar (Colombia). The sample was collected in an 8 L plastic container. The container used for sample Rev. Ambient. Água vol. 15 n. 1, e2448 -Taubaté 2020 collection was pre-treated by washing with alcohol and later rinsed four times with distilled water. The sample was stored at a temperature below 4°C to avoid any physico-chemical changes in the effluent. Finally, the sample volume was divided in order for the pH values to be adjusted to 5.0 and 6.5 by employing HCl solution 0.5 N.

Activation and conservation of Yarrowia lipolytica ATCC 9773
The strain was inoculated at 25°C for three days in petri dishes containing PDA agar ( Figure 1a) and olive oil as a lipid source. A microscopic morphology (Figure 1b) was then carried out employing lactophenol blue as colorant. Lastly, 0.5 mL of the inoculum was adjusted by turbidimetry at MacFarlan scale (3) and stored at 4°C until use.

Preparation of the inoculum and obtaining the crude enzymatic extract (CEE)
The inoculum was obtained from a suspension of mature spores of Y. lipolytica cultivated for five days at 25°C in PDA agar supplemented with olive oil. The biomass obtained was suspended in a solution of 0.9% (w/v) sodium chloride (NaCl). Subsequently, 200 mL of culture medium containing salt water (30% SW), sodium chloride (5.0%), yeast extract (0.5%), olive oil (1.0%) and Triton X-100 (0.1%) were inoculated with a suspension of Y. lipolytica for an incubation time of 8 hours. The fungal biomass was then separated from the supernatant by centrifugation at 5000 rpm for 10 min. Finally, the supernatant [enzymatic extract (EE)], was filtered through cellulose acetate membranes (0.22 to 0.45 mm) and the suspension cell viability was determined by spectrophotometry (Spectronic 20D) at 600 nm (Wu et al., 2009).

Physicochemical effluent characterization
The physicochemical analyses were performed using the Standard Methods protocols established for raw water and wastewater. The fat and oil content was determined by the Soxhlet method according to the Standard Methods of the APHA et al. (2012). The hardness was measured by titration using EDTA solution as titrating agent, the results were expressed as mg of CaCO3/L (Method 2340 C). Biological oxygen demand (BOD) was estimated by preparing the required volume of dilution water with the addition of nutrients and incubating for a period of five days at 20°C, while chemical oxygen demand (COD) was determined based on the rapid dichromate oxidation method (APHA et al., 2012). The phosphorus content was determined by acid digestion, using the ascorbic acid method expressed in mg of P/L. Protein content was determined by the Kjeldahl method. Hydrogen potential was determined potentiometrically using a digital potentiometer (Bench pH-Conductivity meter PC 510).

Evaluation of biodegradation
This test was performed during the effluent's discontinuous fermentation using different concentrations (8%, 12% and 16%) of CEE of Y. lipolytica, considering the volume of the effluent's residual fat (pH 5 and 6.5 at 25°C). Fat content was determined each 8 hours until reaching 32 hours of fermentation. It is noteworthy to mention that BOD5 and COD were calculated only for that time where the best fat removal was reached (inoculum concentration: 16%, incubation time: 32h and pH: 5.0).

Statistical analysis
The percentage of fat removal was used as the response variable. These data were analyzed by means of analysis of variance (ANOVA one way) in order to determine statistically significant differences (P <0.05) between the samples. SPSS software (Version 17.0 for Windows) and the multiple comparison test of Tukey were used. All tests were completed in triplicate.

Growth of Y. lipolytica
The growth curve of Y. lipolytica ATCC 9773 under experimental conditions is illustrated in Figure 2a y 2b. These conditions were chosen from previous analyses of the research group. The figures show that there were mainly three phases: (1) lag phase, which lasted from 0 to 8 h, and the absorbance was 0.332 (pH 5) and 0.044 (pH 6.5); (2) logarithmic phase, which lasted from 8 to 36 h, and the absorbance increased from 0.332 to 1.319 (pH 5) and 0.044 to 0.412 (pH 6.5); and (3) death phase, the absorbance decreased after 42 h. It is interesting to mention that no stationary phase was observed, since the fermentation process was stopped at 44 h, which was before the appearance of the stationary phase.
This experimental result showed that Y. lipolytica was capable of using oil salts as the sole source of carbon, nitrogen and energy, and Y. lipolytica presented remarkable growth in oil and salt wastewater.  According to the results of the BOD5 and COD analyses, the dairy residue can be considered highly biodegradable. Y lipolytica ATCC 9773 is able to reduce BOD5 43.32% and COD 44.30%. The best result in fat removal were obtained when a pH of 5 and an inoculum concentration of 16% were used. It seems that a pH increase in the effluent has effects on Y. lipolytica which influence its growth and therefore the lipases production.
One-way ANOVA of BOD5 and COD values revealed significant differences (p<0.05) among the studied samples when the effluents' pH increased. Regarding to the inoculum concentration, also significant differences (p<0.05) were found at pH 5.0 when inoculum increased; however, the same was not observed at pH 6.5. Similar results have been reported by other researchers (Wu et al., 2009;Wu and Wan, 2008) due to the fact that a high inoculum concentration can encourage microbial growth. Studies involving considerable reduction in COD and BOD in wastewater by using bacterial isolates have been published by Das and Santra (2010), Gaikwad et al. (2014). As with most other agro-industries, the dairy industry produces strong wastewaters characterized by COD and high BOD absorptions signifying their elevated organic content (Orhon et al., 1993). The drop in COD values may be due to the concentration of nutrients, which microbial cultures could use for growing. Current results are in accordance with the reduction in COD reported by Guillen-Jimenez et al. (2000), where maximum COD fall was found up to 65-70%. Similar decrease in COD of the dairy wastewater (99.9%) was noted by Cosa and Okoh (2014) with a consortium of two marine species.
When the pH is 5.0 and the inoculum concentration 16%, the total solids removal reached values close to 13.58%. These results are opposite those reported by Stefańsk et al. (2018) who reported similar removal values employing a pH of 6.5 and an inoculum concentration of 8%. On the other hand, the hardness values are slightly higher than those initial values; this increase could be caused by the production of solid materials generated by the microbial growth. Figure 3 shows the values of fat removal at different concentrations of inoculum (8%, 12% and 16%) in the effluent, adjusted at two pH values (5.0 and 6.5). To pH 5.0 the highest fat removal values (88. 82%) were achieved using an inoculum concentration of 16% and 32 h fermentation time. No significant differences were found (p>0.05) among different inoculum concentrations at the same fermentation times. It should be mentioned that significant differences (p<0.05) were found in fat biodegradation for all fermentation times.
The fermentative process at a pH of 6.5 using different inoculum concentrations is illustrated in Figure 3, where a removal percentage of 49.63% after 8 hours of fermentation may be observed. When the inoculum amount was increased, no significant differences (P>0.05) were appreciated in the fat removal percentage for different samples in the same fermentation time. After 8 hours of fermentation, the percentage of fat removal remained constant, although Y. lipolytica ATCC 9773 continues to present lipase activity, which results in a decreasing of fat percentage until 32 hours of fermentation time is reached. It must be highlighted that significant differences are found (p <0.05) in the percentages of biodegradation obtained at higher fermentation times. At 8% inoculum concentration, more than 16% fat biodegradation was obtained during the same fermentation time (32 hours). It seems that an increase in cell concentration causes cyanic changes in the microorganism, which result in a low metabolic activity, resulting in a reduction of lipase activity. On the other hand, no significant differences were found (p>0.05) between inoculum concentrations of 12% and 16%.
The values of fat removal at different pH concentrations and fermentation times are shown in Table 2. The highest fat removal value (82.88) was obtained at pH 5 using an inoculum concentration of 16%, while the lowest value (47.50) was reached at pH 5 and an inoculum concentration of 8%. It should be highlighted that significant differences (p<0.05) were found for all the fermentation times.