Selection of filamentous fungi producing lipases from residual waters of slaughterhouses

1 Center of Exact and Environmental Sciences Postgraduate Program in Technology and Innovation Management PPGTI Regional Community University of Chapecó UNOCHAPECÓ Av. Senador Attílio Fontana, 591-E EFAPI 89809-000 Chapecó Santa Catarina, Brazil. 2 Paraná Southwest Education Union UNISEP Course in Environmental Engineering Av. Presidente Kennedy, 2601 Dois Vizinhos Paraná Brazil. 3 Federal University of Pelotas – UFPel Postgraduate Program in Materials Science and Engineering. Rua Flores da Cunha, 809 – Pelotas – Rio Grande do Sul, Brazil.


INTRODUCTION
Industrial processes are one of the major factors responsible for water pollution and contamination through the untreated release of effluents into natural waterways, causing significant damage to the environment and the population.One of the main sources of agro-industrial waste that needs special attention in order to avoid the *Corresponding author.E-mail: beckerside@unochapeco.edu.br.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License pollution of the waters, are the effluents from slaughterhouses (Cammarota and Freire, 2006).The undue release of slaughterhouse effluents causes changes in the characteristics of the water and soil, and can pollute or contaminate the environment (Mees, 2004;Chaubey et al., 2013;Miranda and Souza, 2015).
Slaughterhouses represent an important sector in the food industry from the economic point of view.However, considering the large number of companies that still dispose of their effluent without any kind of treatment into the waterways, the contribution of these industries to water pollution is quite significant.Most of these industries are not qualified to deal with the changes required for the implementation of clean technologies, nor for the operation of effluent treatment systems (Vazoller et al., 1991;Machado, 1999;Klibanov, 2001;Kopec et al., 2016).The waste water from slaughterhouses has a high flow rate and high load of suspended solids, organic nitrogen and a BOD 5 of 4,200 mg/L (Aguilar, 2002), depending on the reuse or treatment of the effluent.Because of its composition, this waste is highly putrescible and starts to decompose in a few hours, forming gases that produce bad odors that make it difficult to breathe in the establishment's surroundings, causing nuisance to the local population.These effluents are therefore responsible for the bad image that the public has of these establishments.
Slaughterhouses generally release their effluents, treated or not, into bodies of water.Resolution no.357 of the National Environmental Council (CONAMA) of March 17, 2005, established the classification of water bodies and environmental guidelines for this framework, in addition to the conditions and standards of effluent discharge.Article 34 of this resolution states the conditions and standards for the release of effluents, including a pH between 5 and 9, up to 1 mL/L of sedimentary material, in a test of 1 h in a Imhoff cone, up to 20 mg/L of mineral oils, and up to 50 mg/L of vegetable oils and animal fats (Conama, 2006).
The lipids present in these effluents cause flotation of the biomass and poor formation of sludge granules in anaerobic reactors with upward flow (Hanaki, 1981;Gavala, 1999); toxicity to acetogenic and methanogenic microorganisms (Hanaki, 1981); foaming due to the accumulation of non-biodegraded fatty acids (Vazollér et al., 1991) and a decrease in the concentration of adenosine triphosphate (ATP) (Hanaki, 1981;Perle, 1995;Manoel et al., 2015;Melais et al., 2016;Pecha et al., 2016).The anaerobic digestion of these residual waters rich in fat of animal origin is therefore a slow process, with the release of fatty acids by specific microorganisms with lipolytic activity being a limiting factor (Perle, 1995;Pecha et al., 2016).
The use of lipases promotes an increased reaction speed in the sequential hydrolysis of lipids, reducing the biodegradation time of these compounds (Mendes and Castro, 2004).Lipases are enzymes of animal, plant or microbial origin classified as glycerol ester hydrolases (E.C. 3.1.1.3).They act in the organic/aqueous interface, catalyzing the hydrolysis of the ester-carboxylic bonds present in acylglycerols with the release of fatty acids and glycerol (Yahya, 1998;Smaniotto et al., 2012;Melais et al., 2016;Pecha et al., 2016).The use of microbial lipases is more appropriate for the pre-treatment of industrial waste water rich in animal fats.These lipases are more efficient in the hydrolysis of triacylglycerols containing fatty acids with more than 12 carbons (Masse et al., 2001;Wu et al., 2014;Pecha et al., 2016).
The microorganisms hydrolyze the triacylglycerides in the extracellular medium through the action of lipases, producing fatty acids and glycerol.The lipases (triacylglycerol acyl hydrolases, E.C.3.1.1.3)comprise a group of hydrolytic enzymes that act on the organicaqueous interface, catalyzing the hydrolysis of estercarboxylic bonds, present in glycerides.These enzymes cleave specific triglycerides, but may not be completely specific and catalyze the hydrolysis reactions of triacylglycerides that contain different fatty acids in their composition, which may be of great interest for the treatment of effluents with high fat content (Akesson et al., 1983;Quilles et al., 2015;Pecha et al., 2016).
Within this context, alternative processes are being used for the reduction of the concentration of lipids in these effluents through the action of enzymes and microbial lipases, in particular.These enzymes are of particular importance because they specifically hydrolyze fats and oils, which can be of great interest for the treatment of effluents with high fat content (Iwai and Tsujisaka, 1974).Their use reduces the levels of suspended solids and lipids, enabling better operating conditions in the anaerobic treatment and freeing up piping from oily films, increasing the useful life of equipment (Brockerhoff and Jensen, 1974;Arnold et al., 1975;Hasan et al., 2006;Gupta et al., 2012;Chen et al., 2012;Nascimento et al., 2013;Silalahi et al., 2014).The objective of this work was therefore to isolate microorganisms with the potential of producing lipases, which could be used in waste water treatment systems.

Collection of biological material
Waste water samples were collected from the treatment lagoons of a slaughterhouse located in the municipality of Francisco Beltrão in the State of Parana, Brazil (Figure 1), at the geographical coordinates with Latitude: 26° 02' 54" S and Longitude: 53 03' 74" W. The waste water was collected in cleaned and sterilized plastic containers in order to conserve its characteristics.After collection, the material was transported to the laboratory and stored in a refrigerator at 0°C.All the activities were performed in the Laboratory of Microbiology of the União de Ensino do Sudoeste do Paraná -Unisep -Francisco Beltrão Campus -Paraná, Brazil.

Total microorganism count
For the isolation and total count of microorganisms (filamentous fungi, yeasts and bacteria), a PCA (Plate Count Agar) culture medium was used containing 1% of casein peptone; 2.5% of yeast extract, 2.5% of glucose, 2% of agar.The pH was adjusted to 6.5 and autoclaved at 1 atm of pressure, for a time of 30 min.The effluent samples collected from the lagoons were submitted to successive dilutions in test tubes.The material of each tube was homogenized and inoculated with the pour plate method by adding 1.0 mL of each tube to the bottom of the Petri dish.The plates were subsequently submitted to slow agitation, kept until cooling, incubated upside down at a temperature of 28°C, and monitored every 24 h for the total count.
Based on the microbial growth, each colony characteristic of filamentous fungi was transferred to the culture medium containing 5% fat of animal origin, in order to isolate microorganisms with the ability to grow in a fat rich medium, and incubated at 28°C and monitored every 24 h.After the growth, the isolated and purified colonies were subcultured in tubes containing medium, tilted for keeping, at a temperature of ± -4°C for later identification and assessment of their capacity to produce lipases.The fungal colonies that were distinct from one another, according to macroscopic observations (color and growth features in the culture medium) were purified in PDA medium, preserved by subculture and stored at 4°C.To identify the genera, microcultures from the selected isolates were made using agar block, and literature was consulted (Booth, 1971;Barnett and Hunter, 1972;Larone, 1993;Menezes and Oliveira, 1993).

Assessment of enzyme activity
The isolated and purified colonies of filamentous fungi were evaluated in order to obtain lipase producing strains.The methodology described by Hankin and Anagnostakis (1975) was followed to perform this activity.The culture medium used for this activity was composed of: peptone (1%), sodium chloride (0.5%), calcium chloride (0.01%) and agar (2%).To this medium, 1% sorbitan monolaurate was added (Tween-80  ).After 72 h of incubation at 28ºC, the Petri dishes were kept in the refrigerator for 7 days.As such, lipase activity was observed through the presence of calcium salt crystals of the lauric acid, released by the enzyme around the formed colonies.Each colony was measured with a caliper and the enzymatic index was determined using the following Table 1.Total count of microbial flora present in the slaughterhouse effluent.

Micro-organism
Count in CFU mL  ratio: EI = diameter of the colony + the halo / diameter of the colony.

Statistical analysis
The statistical analysis was performed using the software Statistica , version 5.0.The analyses of variance were carried out according to standards of ANOVA.The significant differences between the means were determined by Tukey's test.All activities were performed in triplicate.

RESULTS AND DISCUSSION
After the development of the work, the microbial flora of the effluent collected in the treatment lagoons of the evaluated slaughterhouses could be quantified.The results are summarized in Table 1.By analyzing Table 1, one can see that bacteria are the predominant microorganisms in the slaughterhosue effluent, with 6.8 × 10 8 CFU.mL -1 , followed by filamentous fungi with 3.4 × 10 4 CFU.mL -1 and yeasts with 2.2 × 10 2 CFU.mL -1 . This diversity can be seen as beneficial for the treatment process of these effluents because a greater diversity increases the biodegradation capacity of the effluents (Pereira, 2007;Bhuwal et al., 2013;Motiwalla et al., 2013).
The isolated, purified and identified fungi were: Aspergillus flavus (L 1 ), Paecilomyces sp (L 2 ), Aspergillus terreus (L 3 ), Penicillium chrysogenum (L4) and Cladosporium herbarum (L 5 ) and Aspergillus niger (L 6 ) (Figure 2).After isolation and identification, the fungi were evaluated for their ability to degrade lipids.The  results obtained are summarized in Table 2 and in Figures 3. The lipase activity was observed through the presence of calcium salt crystals from lauric acid, released by the enzyme around the formed colonies, revealing the degradation of the sorbitan monolaurate (Tween-80  ) (Figure 3).This behavior is in line with the work by Sharma et al. (2001), Maia et al. (2001), Dominguez et al. (2003), Rodríguez-Contreras et al. (2012a), Rodríguez-Contreras et al., (2012b), Sandhya et al., (2013) and Vinish et al. (2015), who assessed the production of lipases using the same methodology of this work and noted that two of the tested strains showed the same precipitation pattern of calcium laurate forming a thin layer of precipitation around the colonies, which may also be a coarse grain precipitation.
After analyzing the lipolytic activity, no differences were found with respect to the production of lipases between the strains L 1 of A. flavus and L 3 of A. terreus, with enzymatic indices of 2.02 and 2.10, respectively.Another group of strains that did not show any differences between themselves regarding the enzymatic indices, were the strains L2 of Paecilomyces sp, L 4 of P. chrysogenum and L 5 of C. herbarum, presenting rates of 1.49, 1.66 and 1.51, respectively.The strain with the lowest enzymatic index, and which stood out from the other strains, was the L 6 strain of A. niger, with an enzymatic index of 1.36.After evaluation of the obtained indices, it was found that the fungi that produce the most enzymes were the fungi of the genus Aspergillus, with the species A. flavus and A. terreus.
The results obtained are in agreement with the results found in Soza-Gomes and Alves (1983), who found enzymatic indices between 1.45 and 1.86 evaluating strains of fungi with potential to produce extracellular enzymes.They also found enzymatic indices equal to 1 (one), indicating that these strains showed no lipolytic activity, at least not extracellularly.The fact that the fungal strains L2, L4, L5 and L6 have shown low enzymatic indices for the production of lipases may be related to the phenotypic responses to enzyme induction mechanisms (Fargues and Robert, 1983;Amirita et al., 2012;Lutz et al., 2013;Senanayake et al., 2016) or to the culture medium used.However, with respect to the type of medium used for the observation of lipolytic activity, it is important to emphasize the fact that fungi may have different responses to the composition of fatty acids used as source of lipids.Gabriel (1968) and Silva et al. (2015), observed that Metarhizium anisopliae and Beauveria bassiana continually degrade lipids in a specific medium to detect lipase, but they do not produce enzymes capable of degrading olive oil or coconut oil, suggesting that this selective reaction could be related to the presence of different fatty acids, such as oleic acid in olive oil, and capric and caprylic acid in coconut oil.The lipolytic activity in different fungi may therefore be related to the species-specificity of the fungus to the substrate.As such, one can see that the medium containing Tween-80 may not be the most appropriate for the evaluation of lipolytic activity, since this activity may be related to other types of fatty acids.

Conclusion
The results obtained in this study enabled the conclusion that the waste water produced in slaughterhouses has a high microbial load, highlighting bacteria with 6.8 × 10 .Out of this microbial population, six species of lipase producing fungi were isolated, with the fungi A. flavus and A. terreus being those that obtained the highest enzymatic indices.

Figure 1 .
Figure 1.Waste water collection location from the slaughterhouse.

*
Values followed by the same letter vertically, do not differ significantly at the level of 5% by the Tukey Test.

Figure 3 .
Figure 3. Halo of degradation caused by the lipolytic activity revealing the presence of calcium salt crystals of lauric acid.

Table 2 .
Lipolytic activity of the filamentous fungi strains isolated from the slaughterhouse effluent.Indices followed by the same letter in the column do not differ by Tukey's Test at the level of significance of 5%. #