Molecular, physiological, and biochemical characterization of extracellular lipase production by Aspergillus niger using submerged fermentation

Isolation of fungal strains

Oil seeds of pistachios, almonds, walnuts, cashews, sunflower, sesame, corn, and peanuts were collected from the local markets in different cities of Saudi Arabia (Dammam, Al-Khobar, Al-jubail, Hafr Al-Baten, and Al-Hassa). The seed surfaces were sterilized using 5% sodium hypochloride and dried. The seeds were incubated in Tween 80 agar as described by Malekabadia, Badoei-dalfarda & Karamia (2018) for 7 days at 27 °C. Then samples of grown fungi were purified on potato dextrose medium according to the method described by Ziaee, Zia & Goli (2018) and incubated for 5 days for further examination.

Screening for lipase production by fungal isolates

For the screening of lipolytic fungi, the phenol red and tributyrin plate assays were applied to the isolates obtained from the Tween 80-treated samples. First, lipase activity was evaluated using phenol red agar medium to detect pH changes arising due to the hydrolysis of oil into fatty acids; typically, the pH of the medium reduces upon hydrolysis and is characterized by a yellow halo (Singh et al., 2006). The best isolates for enzymatic production were then selected to evaluate hydrolysis on tributyrin agar (TBA) medium to confirm the productivity of lipase that digests tributyrin, resulting in a colorless region as descried by Ayinla, Ademakinwa & Agboola (2017). Lipase production was determined by measuring the diameter of the yellow and clear zones (halos) that formed around the fungal growth in the phenol red and tributyrin agar media, respectively.

DNA extraction and polymerase chain reaction (PCR)

Genomic DNA of the samples of Aspergillus isolates was extracted using the Wizard® Genomic DNA Purification Kit (Promega, WI, USA), according to the manufacturer’s protocol. Polymerase chain reaction (PCR) amplification reaction was performed in a final volume of 50 µl containing the following final concentrations of each component: 10 × PCR Top Taq Buffer, 25 mM MgCl₂, 10 mM of each deoxynucleoside triphosphate, 2 U Top taq polymerase, and 10 µM each of 18SrRNA F: 5′-GCTTAATTTGACTCAACACGGGA-3′ and 18SrRNA R: 5′-AGCTATCAATCTGTCAATCCTGTC- 3′ primers to amplify 18SrRNA genes. The thermal cycling parameters were as follows: initial denaturation at 95 °C for 10 min, followed by 35 cycles of amplification at 95 °C for 1 min, annealing at 67.7 °C for 75 s, and extension at 72 °C for 2 min, with a final extension step of 5 min at 72 °C. In addition, lipase primers descried previously by Metwally et al. (2015) forward primer: 5′-ATGTTCTCTGGACGGTTTGGAGTG-3′; reverse primer: 5′-TTATAGCAGGCACTCGGAAATC-3′—were used to amplify the lipase gene of A. niger (Table 1). Identical thermal cycling parameters were used for the amplification of the lipase gene, except for the annealing temperature, which was set at 64.7 °C.

PCR purification and sequencing

The PCR product of the 18S rRNA gene was purified using QlAquick PCR purification kit (QIAGEN, Germany), following the manufacturer’s instructions. Purified amplicons were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, USA), with 1 µM primers (either 18SrRNA F or 18SrRNA R) and dideoxynucleotides (ddNTPs). The amplification products were purified using the BigDye X-Terminator purification kit (Applied Biosystems, Thermo Fisher Scientific, USA), according to the manufacturer’s protocol, and sequenced using Genetic Analyzer 3500 (Thermo Fisher Scientific, USA). Initial sequencing analysis was performed using 3500 Data Collection Software (Applied Biosystems, Thermo Fisher Scientific, USA), followed by further analysis using the basic local alignment search tool (BLAST), phymycodb, and MAFFT tools. The generated fasta sequence files were ultimately submitted to NCBI through Genbank.

Random amplification polymorphic DNA (RAPD) analysis

The reactions were performed in 25 µl mixtures containing 25 ng extracted DNA, 1 µM primer, 20 mM Tris–HCl (pH 8.8), 2.5 mM MgCl₂, 0.2 mM of each dNTP, and 1 U Taq DNA polymerase. Five primers were found suitable for RAPD analysis (Table 2) after they were tested in discriminate strain typing of Aspergillus spp. Amplifications were conducted with RAPD1, RAPD2, RAPD3, RAPD4, and RAPD5 primers under the following thermal cycling conditions: initial denaturation for 2 min at 94 °C, followed by 44 cycles of denaturation for 1 min at 94 °C, annealing for 2 min at 36 °C, and extension for 2 min at 72 °C, with a final extension step for 10 min at 72 °C. Amplified products were resolved in a 1.5% agarose gel and detected by ethidium bromide staining. DNA bands were analyzed using the UV-based detection method with the GelDoc XR imager (Bio-Rad Laboratories Inc., Hercules, CA). A 100-bp DNA ladder (Quick-Load® 100 bp DNA Ladder, QIAGEN, Germany) was used to estimate DNA fragment sizes.

Lipase enzyme production by submerged fermentation

Lipase production was examined using Tween 80 broth, as described previously (Ayinla, Ademakinwa & Agboola, 2017). The sterile medium was aseptically inoculated with 2 ml of 3 × 10⁷ spores/ml fungal conidial suspension that was prepared in a mineral salt solution. The culture was incubated at 25 ± 2°C for 5 days in a rotary shaker at 200 rpm. Post-incubation, the fungal broth was filtered using Whatman’s filter paper no. 1, and the top growth was dried in an oven at 60°C prior to weighing. The filtrate was centrifuged at 1,200 rpm for 30 min at 4°C, and the clear supernatant was subjected to the lipase assay using a spectrophotometer (SPECTRO UV–VIS DOUBL).

Lipase enzyme activity assay

Lipase activity was assayed using the p-Nitrophenyl Palmitate (pNPP) hydrolysis method. The filtrate (100 µl) was incubated for 15 min at 30 °C with 800 µl of 0.25% polyvinyl alcohol (PVA) solution (pH: 6.5) and 100 µl of 3 mM pNPP solution in isopropanol. The reaction was terminated by adding 500 µl of 3 M HCl. Next, 500 µl of the centrifuged mixture was added to 1 ml of 2 M NaOH. Absorbance was measured in spectrophotometer (SPECTRO UV–VIS DOUBL) at 410 nm and the unit of the enzymatic activity (U) was determined from the release of 1 µM of pNPP per minute. A standard curve was used to measure enzyme activity, as described previously by Oliveira, Fernandes & Mariano (2014).

Physiological and biochemical characterization

To determine the optimal parameters, cultures were incubated at different temperatures ranging from 15 to 45 °C (Sundar & Kumaresapillai, 2013), various pH levels ranging from 3 to 8.5 (Sethi, Tyagi & Anurag, 2016), various time periods of 3, 5, 7, 10, and 15 days (Niaz et al., 2014), different carbon sources; glucose, fructose, lactose, maltose, galactose, sucrose, and starch (Bindiya & Ramana, 2012), different nitrogen sources; peptone, yeast extract, beef extract, sodium nitrate, casein and potassium nitrate 6 (Ayinla, Ademakinwa & Agboola, 2017), various amounts—0.5, 1, 1.5, 2.5, and 3 ml—of spore suspension (3 × 10⁷ spores/ml) (Niaz et al., 2014), and different lipid sources—olive, sunflower, corn, soybean, palm, and castor oil—were used at various concentrations—1.5, 2.0, and 2.5 ml. Lipase activity was assayed in the filtered culture for all factors using submerged fermentation medium (Reshma & Shanmugam, 2013).

Statistical analysis

Correlations between the enzyme activity and temperature, pH, incubation time, carbon source, nitrogen source, oil type, and inoculum volume were examined by conducting analysis of variance (ANOVA) using SPSS software version 23 (George & Mallery, 2016).

Isolation of fungi from oil seeds

A total of 256 fungal isolates were obtained from culturing the following seeds: pistachio, cashew, almonds, corn, nut, and peanut, while sesame and sunflower seeds did not reveal any fungal growth. The highest number of fungal isolates among oil seeds was observed in pistachio, followed by cashew, almonds, corn, nut, and peanut seeds, in descending order. In addition, Aspergillus sp. were the most frequent isolates (n = 133, 52.00%), followed by Penicillium sp. (n = 63, 24.61%), Alternaria sp. (n = 32, 12.50%), and Fusarium sp. (n = 28, 10.94%, Table 3; Fig. S1).

Screening of lipase production by fungal isolates

From each genus, one isolate was selected from each crop found in any of the five outlined cities (n = 35) to evaluate lipase production: Aspergillus (n = 13 isolates), Penicillium (n = 10 isolates), Alternaria (n = 7 isolates), and Fusarium (n = 5 isolates). Lipase production of the isolates was examined using the phenol red agar medium. Aspergillus spp. were found to be the highest lipase producers among various fungal genera, which produced less to no enzymes (Table S1). Therefore, Aspergillus isolates that were able to hydrolyze olive oil using the phenol red agar medium were selected for further analysis depending on their ability to hydrolyze tributyrin. The clear zones obtained using the tributyrin plate assay (Fig. S3) revealed that A. niger MH079049.1, A. niger MH111400.1, A. niger MH078565.1, A. niger MH078571.1, and A. niger MH111398.1 were the most efficient lipase producers (Table 4).

Lipase enzyme assay

Lipase activity was estimated using the spectrophotometric pNPP hydrolysis assay. Aspergillus isolates A. niger MH079049.1 (406.92 U/ml), A. niger MH078571.1 (390 U/ml), A. niger MH111400.1 (267.69 U/ml), A. niger MH078565.1 (255.38 U/ml), and A. niger MH111398.1 (196.92 U/ml) showed higher lipase activity than other isolates, such as A. niger MH111401.1 (35.38 U/ml), which showed minimal activity (Table 5). In addition, the results indicated no correlation between lipase activity and the growth of Aspergillus isolates.

Molecular characterization of Aspergillus spp. isolates

Isolated Aspergillus spp. were identified using 18srRNA gene sequencing. All 11 Aspergillus strains that were tested in the current study were identified as A. niger. The sequences were submitted to GenBank under the accession numbers MH111398.1, MH111399.1, MH057541.1, MH111400.1, MH078513.1, MH078565.1, MH111401.1, MH078566.1, MH078571.1, MH079049.1, and MH079063.1. In addition, the presence of the lipase gene was verified in all 11 strains by PCR amplification (Fig. S5).

RAPD analysis using five primers to amplify random regions of 11 A. niger strains genomic DNA yielded 285 fragments that could be analyzed. All of the five primers amplified fragments across the 11 studied strains, with the number of amplified fragments varying from two (Fig. 1C) to eight (Fig. 1E) and ranging from 200 bp to 2,500 bp. Of the 285 amplified bands, five were polymorphic with an average of one polymorphic fragment per primer. Polymorphism ranged from 39.77% for (Fig. 1A) to a maximum of 53.63% (Fig. 1B), with an average of 45.99%. Only 1 (Fig. 1B) out of 5 primers showed more than 50% polymorphism. The extent of polymorphism detected among the isolates, as revealed by RAPD, is represented in (Figs. 1A to 1E). A dendrogram based on UPGMA analysis grouped the 11 A. niger strains into main clusters. A1 to B5 and C1 to C4 indicate clusters at the level of 60% and 75% similarity in individual RAPD analysis, respectively (Figs. 2A to 2E); whereas, AC and BC indicate clusters with 75% genetic similarity in the combined analysis of the random polymorphic amplicons (Fig. 2F).

Physiological and biochemical characterization of lipase production in Aspergillus isolates

Five Aspergillus isolates were selected as the highest lipase producers based on the results of the lipase production test using the tributyrin and lipase activity tests. The isolates A. niger MH111398.1, A. niger MH111400.1, A. niger MH078565.1, A. niger MH078571.1, and A. niger MH079049.1 were examined in a submerged fermentation culture (Smf) to measure their enzymatic activity.

Effect of temperature

The metabolic activity at 0 °C is low, which results in the inhibition of lipase activity. At 40 °C, the isolate that exhibited the highest lipase activity was A. niger MH078571.1 (575.9 U/ml), while the A. niger MH111400.1 exhibited the lowest lipase activity (470.51 U/ml). At 45 °C, two out of five isolates were able to grow in the submerged culture–A. niger MH078571.1 (403.3 U/ml) and A. niger MH079049.1 (524.87 U/ml)–whereas, the other isolates were not able to grow under these conditions. However, lipase production at this temperature was lower than that at 40 °C. Therefore, the optimum temperature was fixed at 40 °C (Table S2; Fig. 3A). The measurement of the dry weight of all fungal isolates showed that a temperature of 15 °C was the optimal for fungal growth in the submerged fermentation culture, but it was a poor catalyst for lipase production. In addition, mycelium growth was measured to determine the extent of fungal growth. The mycelium diameter increased as the temperature increased up to 30 °C, and then gradually began to decrease as the temperature further increased.

Effect of pH

The results of the present investigation showed that increasing the acidity of the culture plays a role in inhibiting lipase production and increasing alkalinity; a pH of 7.5 yielded optimal lipase production for the five tested isolates, with the highest lipase activity exhibited by A. niger MH078571.1 (610.77 U/ml) and A. niger MH079049.1 (606.15 U/ml). Most isolates had lost their ability to grow at pH 8.5, except two isolates—A. niger MH078571.1 and A. niger MH079049.1—both of which were able to grow and produce lipase under alkaline conditions of the culture; however, their lipase yields were low. Therefore, a pH of 7.5 was selected as the optimum pH for lipase production for the selected isolates (Table S3; Fig. 3B).

Effect of incubation time

The results in Table S4 and Fig. 3C demonstrate that the optimal duration for the highest lipase production was 3 days. The longer the incubation of the isolates, the lesser the lipase yield. The lipase activity of the isolates reached minimum values after 15 days of incubation. For instance, A. niger MH111398.1 showed low lipase activity (348.21 U/ml) after 15 days. In contrast, mycelium growth density in the submerged culture as well as on surface of the petri dishes increased with an increase in the incubation duration; the isolate yielding the highest density after an incubation period of 15 days was the dry mycelium of A. niger MH079049.1 (1.246 g). Therefore, the duration of incubation was fixed to study the physiological factors after 3 days of incubation.

Effect of carbon source

Five fungal isolates were grown on media containing olive oil as the sole source of energy without the presence of a carbon source to stimulate lipase production. However, the presence of a carbon source contributes to improvement of fermentation and subsequently increases cellular metabolism. Thus, increasing cellular metabolism contributes to increased fungal lipase activity. Different carbohydrates acted as monovalent sources for optimal enzymatic production; fungi produce low levels of lipase in the presence of starch and sucrose. Fructose exhibited the highest positive impact on lipase production in A. niger MH078571.1 (717.44 U/ml), followed by A. niger MH079049.1 (710.26 U/ml) (Table S5; Fig. 3D). These results showed a correlation between mycelium growth and lipase production in the presence of fructose.

Effect of nitrogen source

Yeast extract was shown to be the most effective nitrogen source, and the highest value of 741.54 U/ml was obtained for A. niger MH078571.1, followed by A. niger MH079049.1 (734.36 U/ml). The results outlined in Table S6 and Fig. 3E show that mycelium growth and linear fungal growth were the highest in the presence of peptone; the highest linear growth value was 7.6 cm for A. niger MH078571.1 and the highest mycelium growth value was 1.309 g for A. niger MH079049.1. Casein and potassium nitrate exhibited the least effect on mycelium growth and linear growth.

Effect of inoculum volume

Various amounts—0.5, 1, 1.5, 2.5, and 3 ml—of spore suspension (3 × 10⁷ spores/ml) inoculated on the submerged fermentation culture revealed that the lowest values of lipase production were with an inoculum volume of 0.5 ml for all isolates. On the other hand, lipase production increased as the inoculum volume increased, until reaching the highest value at 2.5 ml; (794.62 U/ml) obtained for A. niger MH078571.1. Inoculum volumes higher than 2.5 ml decreased both lipase production and mycelium growth (Table S7; Fig. 3F).

Effect of different lipid sources

The examination of different oil sources, such as olive, sunflower, corn, soybean, palm, and castor oils at volumes of 1.5, 2.0, and 2.5 ml on lipase activities using the submerged fermentation medium revealed a 2.0 ml/L of palm oil as the optimal oil type and concentration for lipase production (Table S8). The optimal enzymatic production for all of the oil types (olive, sunflower, castor, and palm oils) was at 2% and the increase in the concentration up to 2.5% showed decreased lipase production in all of the tested strains, except for corn oil, which showed optimal production at 2.5%.