Investigation of Lipolytic-Secreting Bacteria from an Artificially Polluted Soil Using a Modified Culture Method and Optimization of Their Lipase Production

Compared to lipases from plants or animals, microbial lipases play a vital role in different industrial applications and biotechnological perspectives due to their high stability and cost-effectiveness. Therefore, numerous lipase producers have been investigated in a variety of environments in the presence of lipidic carbon and organic nitrogen sources. As a step in the development of cultivating the unculturable functional bacteria in this study, the forest soil collected from the surrounding plant roots was used to create an artificially contaminated environment for lipase-producing bacterial isolation. The ten strongest active bacterial strains were tested in an enzyme assay supplemented with metal ions such as Ca2+, Zn2+, Cu2+, Fe2+, Mg2+, K+, Co2+, Mn2+, and Sn2+ to determine bacterial tolerance and the effect of these metal ions on enzyme activity. Lipolytic bacteria in this study tended to grow and achieved a high lipase activity at temperatures of 35–40 °C and at pH 6–7, reaching a peak of 480 U/mL and 420 U/mL produced by Lysinibacillus PL33 and Lysinibacillus PL35, respectively. These potential lipase-producing bacteria are excellent candidates for large-scale applications in the future.

The effect of water content and carbon source addition on lipase production from Aspergillus aculeatus Ms.11 using spent coffee ground

Lipase is one of the industrially important enzymes, however, the production needs costly substrate. To overcome the problem, we developed an effective medium formulation to produce lipase from indigenous lipolytic mould Aspergillus aculeatus Ms.11 using spent coffee ground. We observed the effect of additional glucose and olive oil, and the influence of water content on lipase production. The experiments were done using the Solid-State Fermentation (SSF) method for 7 days. The results show that optimum lipase production on substrates with additional glucose and olive oil observed on day 4 with lipase activity of 16.296 U/mL and lipase productivity of 150.32 U/g/day. The optimum water content from the results is 50%. The highest lipase activity obtained using the water content is 291.80 U/mL, while the highest lipase productivity is 106.32 U/g/day. The results showed that water content as well as the addition of glucose and olive oil, affects lipase productivity of Aspergillus aculeatus Ms.11 on spent coffee ground. Further studies to optimise the production condition are suggested.

Optimization of lipase production using fungal isolates from oily residues

Lipases are triacylglycerol hydrolases that catalyze hydrolysis, esterification, interesterification, and transesterification reactions. These enzymes are targets of several industrial and biotech applications, such as catalysts, detergent production, food, biofuels, wastewater treatment, and others. Microbial enzymes are preferable for large scale production due to ease of production and extraction. Several studies have reported that lipases from filamentous fungi are predominantly extracellular and highly active. However, there are many factors that interfere with enzyme production (pH, temperature, medium composition, agitation, aeration, inducer type, and concentration, etc.), making control difficult and burdening the process. This work aimed to optimize the lipase production of four fungal isolates from oily residues (Penicillium sp., Aspergillus niger, Aspergillus sp., and Aspergillus sp.). The lipase-producing fungi isolates were morphologically characterized by optical and scanning electron microscopy. The optimal lipase production time curve was previously determined, and the response variable used was the amount of total protein in the medium after cultivation by submerged fermentation. A complete factorial design 32 was performed, evaluating the temperatures (28 °C, 32 °C, and 36 °C) and soybean oil inducer concentration (2%, 6%, and 10%). Each lipase-producing isolate reacted differently to the conditions tested, the Aspergillus sp. F18 reached maximum lipase production, compared to others, under conditions of 32 °C and 2% of oil with a yield of 11,007 (µg mL−1). Penicillium sp. F04 achieved better results at 36 °C and 6% oil, although for Aspergillus niger F16 was at 36 °C and 10% oil and Aspergillus sp. F21 at 32 °C and 2% oil. These results show that microorganisms isolated from oily residues derived from environmental sanitation can be a promising alternative for the large-scale production of lipases. Graphical Abstract

Enzyme production by soilborne fungal strains of Aspergillus niger isolated from different localities affected by mining

The research focused on the enzyme production/activity of sixteen Aspergillus niger strains isolated from different localities. The soils vary mainly in their pH value, which ranges from an ultra-acidic (< 3.5) to a very strongly alkaline (> 9.0) environment. Contamination caused by several centuries of mining activities at old mining sites persists at all the localities. The concentrations of toxic elements, such as As, Sb, Zn, Cu and Pb, very often exceed the common limit values. Presence of toxic elements in contaminated sites may affect microscopic fungi and cause changes of their physiology, including the production of different metabolites, such as enzymes. Production of esterase, cellulase, lipase and protease was investigated. Changes in physiological properties, such as the growth and enzymatic activity of the sixteen A. niger wild type strains, were determined. Esterase, cellulase and lipase activity was not determined in the sixteen strains tested. Considerable differences were recorded in the size of the colonies also within strains cultivated on the same nutrient medium. The control strain from locality Gabčíkovo formed the smallest colonies when tracking LA, EA and PA compared with the other strains. Lipase production was determined for several strains at different intensities and was highest in the strain isolated from the uncontaminated locality Gabčíkovo. The enzymatic activity of the other strains isolated from contaminated sites was very low. The achieved results confirmed the direct influence of environmental factors on the physiological properties of the studied strains of Aspergillus niger.

Application and characterization of crude fungal lipases used to degrade fat and oil wastes

Aspergillus niger MH078571.1 and A. niger MH079049.1 were identified previously as the two highest Aspergillus niger strains producing lipase. Biochemical characterizations of lipase activity and stability for these two strains were examined and revealed that the optimal temperature is 45 °C at pH 8for A. niger MH078571.1 and 55 °C for MH079049.1. The lipase production of both strains was studied on medium contains waste oil, as a cheap source to reduce the industrial cost, showed that the optimal incubation period for the enzyme production is 3 days. Moreover, an experiment on lipase activates in organic solvents demonstrated that 50% of acetone is the best solvent for the two strains. In the presence of surfactants, 0.1% of tween 80 surfactant showed the best lipase activities. Furthermore, Mg2+ and Zn2+ ions enhanced the lipase activity of A. niger MH078571.1, while Na2+ and Cu2+ enhanced the enzyme activity of A. niger MH079049.1. Lipase activity was also tested for industrial applications such as integrating it with different detergents. Maximum lipase activity was obtained with 1% of Omo as a powder detergent for both strains. In liquid detergent, 0.1% of Fairy showed maximum lipase activity in A. niger MH078571.1, while the lipase in A. niger MH079049.1 was more effective in 1% of Lux. Moreover, the degradation of natural animal fat with crude enzyme was tested using chicken and sheep fats. The results showed that more than 90% of fats degraded after 5 days of the incubation period.

Purification and characterization of thermo-alkaline stable lipase from Bacillus coagulans and its compatibility with commercially available detergents

Thermo-alkaline stable lipase producing B. coagulans was isolated and identified by 16S rRNA sequencing. The lipase production was optimized using different growth parameters. The enzyme was purified and characterized in terms of pH, temperature, solvents, heavy metal ions and inhibitors. Compatibility with commercially available detergents was also studied. The isolate showed maximum lipase production at 37 ⁰C; pH of 9 within 48 h. Addition of magnesium chloride increased lipase production. Sephadex G-100 chromatography was used to purify lipase. The enzyme showed maximum activity at pH 8 of 30 ⁰C. Lipase form B. coagulans was active at a wide range of temperature between 30-70 ⁰C. It was stable in most of the solvents at a concentration 5 and 10%, except dimethyl sulfoxide (DMSO) and methanol. Iodoacetic acid (IAA) and p-chloromercuribenzoic acid (pCMB) had an inhibitory effect on lipase. The lipase was compatible with commercially available detergents that increased the brightness and whiteness of the tested cotton fabrics. Lipase from B. coagulans with alkaline stability at a wide range of temperature has potential application in the detergent industry.

Wastewater from the Edible Oil Industry as a Potential Source of Lipase- and Surfactant-Producing Actinobacteria

Wastewaters generated from various stages of edible oil production in a canola processing facility were collected with the aim of determining the presence of lipase-producing actinobacteria of potential industrial significance. The high chemical oxygen demand (COD) readings (up to 86,700 mg L−1 in some samples) indicated that the wastewater exhibited the nutritional potential to support bacterial growth. A novel approach was developed for the isolation of metagenomic DNA from the oil-rich wastewater samples. Microbiota analysis of the buffer tank and refinery condensate tank wastewater samples showed a dominance of Cutibacterium acnes subsp. defendens, followed by a limited number of other actinobacterial genera, indicating the presence of a highly specialized actinobacterial population. Cultured isolates with typical actinobacterial morphology were analyzed for their ability to produce lipases and biosurfactants. Two strains, designated as BT3 and BT4, exhibited the highest lipase production levels when grown in the presence of tributyrin and olive oil (1.39 U mg−1 crude protein and 0.8 U mg−1 crude protein, respectively) and were subsequently definitively identified by genome sequencing to be related to Streptomyces albidoflavus. Cultivation of the strains in media containing different types of oils did not markedly increase the level of enzyme production, with the exception of strain BT4 (1.0 U mg−1 crude protein in the presence of peanut oil). Genome sequencing of the two strains, BT3 and BT4, revealed the presence of a range of lipase and esterase genes that may be involved in the production of the enzymes detected in this study. The presence of gene clusters involved in the production of biosurfactants were also detected, notably moreso in strain BT3 than BT4.