3.1. Isolation of yeasts from industrial effluents and contaminated ground water.
The industrial effluent collected from the Bengaluru and contaminated ground water from Gajapathi area in Odisha, lead to the isolation, growth and axenic culturing of one and two yeast colonies respectively. The isolates were coded as BANG3, ODBG2, and ODBG4 for further studies and molecular identification. The microscopic observation exhibited the single cell structure ovoid in shapes with various budding stages along with clear and distinguished cell wall structure. Figure 01 represents the macroscopic and microscopic views of the yeast isolates. There were not much changes in the morphological and microscopic observations, thus the exact identification the isolates was not revealed, and indicated the necessity of molecular based identification.
3.2. Determination of MIC (Minimum inhibitory concentration) /Metal Tolerance Test.
The metal tolerance test/MIC revealed the capability of the isolate ODBG2 later identified as Candida parapsilosis strain ODBG2, to utilize the heavy metals for its growth and tolerated up to the concentration of 2.0mM Pb, 1.5mM Cd and 0.5mM Cr. The significant difference in the growth pattern was observed after 48hr of metal-isolate interaction with respect to Cd and Pb. While significant difference was recorded after 24hr under hexavalent Cr stress. In the second isolate BANG3; Candida sp. strain BANG3; exhibited the metal tolerance up to 2mM of Pb and 1.0mM Cd, with significant growth difference with respect to control (without HM amended) was observed after the incubation period of 72hr and 24hr respectively, whereas zero tolerance towards Cr(VI). The isolate ODBG4; Candida vishwanathii strain ODBG4 was adaptive to grow under Pb at 1.5mM followed by Cr and Cd at 1.0mM with a significant difference after the time interval of 48hr. The growth curve of yeast isolate ODBG2, BANG3 and ODBG4 with and without metal amended is shown in the Fig. 02. The recent study by Bansal et al. (2019) on Candida parapsilosis, an environmental isolate and its metal tolerance have indicated the survival capability and utilization under heavy metal such as Ni, Pb and Cd at the concentration of 12mM. Similarly, the various researches have shown the ability of Candida sp. to tolerate and to grow under various heavy metals which were isolated from contaminated sources like soil, water and industrial effluent system. The difference in metal tolerance might be ascribed to the level of HM contamination in the water samples, the contact or incubation time (Azcon et al. 2010; Munner et al. 2013; Moreno et al. 2019) as well as dependent on metal ion status and species (Balsalobre et al. 2003).The MICs of different isolates has been summarized in the Table 01.
3.3. Molecular Identification of heavy meal tolerant yeast isolates/strains.
The PCR amplicons of the isolates with the codes ODBG2, BANG3, and ODBG4 resulted in the product of ~ 500bp size (Fig. 3). Further the sequencing of the region ITS1, 5.8rRNA and ITS2 using ITS 1 (forward primer) of the amplicons ODBG2, BANG3, and ODBG4 resulted in 493bp, 438bp, and 447bp product. BLAST analysis showed > 98% identity with sequences of Candida parapsilosis (KX652405, JN989529, MNT33072) with respect to our isolate code ODBG2, Candida sp. (MH802509, MN124747, KJ706734) with respective isolate code BANG3 and Candida vishwanathii (KU729067, MK394124,KC608220) with respect isolate code ODBG4. The obtained and identified sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and Accession numbers were obtained. The code of isolates and accession no. has been represented in the table 02.
The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei, 1993). Evolutionary analyses was conducted in MEGA X (Felsenstein, 1985; Kumar et al., 2018), Fig. 4 represence the phylogenetic tree.
3.4. Oxidative stress-induced enzymatic and non-enzymatic studies (SOD, CAT and GSH).
3.4.1 Effect of heavy metal-induced oxidative stress on SOD activity
The primary defense antioxidant enzymes in any biological system are attributed towards the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidases (PO). The activity of SOD was detected based on the percentage inhibition of quercertin autoxidation in the control and treated samples. The SOD activity in Candida parapsilosis strain ODBG2 was found to be high in the range of 16% (Cd 0.5mM), 27.5% (Cd 1.0mM), 22.5% (Pb 0.5mM) and 25% (Pb 1.0mM) during first 24hr of stress, however the gradual decrease in the abundance at 48hr and thereafter (Fig. 5A). The SOD activity in Candida viswanathii strain ODBG4 was relatively high for Cd (0.5mM concentration) at 24hr, for Pb (1mM) it slightly increased at 48 and 72hr (Fig. 5B), indicating minimal effect on SOD. SOD is the primary defense enzyme found to be active during abiotic stress-induced intracellular ROS in biological systems, which interacts and converts into H2O2 and water molecule, leading to the sequential activation of other primary defense enzymes such as CAT and PO, subsequently the non-enzymatic small compounds like GSH (Bandyopadhyay et al., 1999). The varied response of SOD with respective different time intervals and concentrations has been observed in fungi such as Pleurotus ostretus HAU-2 upon Pb (Zhang et al., 2016), Trichosporon asahii upon different heavy metal and metalloid treatments (Ilyas et al., 2014). SOD was found to be not effective at the higher concentration rate and incubation time, which is indicating the role of CAT and PO in the system.
3.4.2. Effect of heavy metals induced oxidative stress enzyme – CAT
The CAT showed increased activity of 38 and 40 µmoles/mg of protein/min upon Pb stress at 05mM and 1.0mM concentration during at 48hr incubation, subsequently decreased on 72hr of incubation with 20 and 28 µmoles mg of protein/min in Candida parapsilosis strain ODBG2 (Fig. 6A). Similarly, CAT activity was higher at 48hr under Pb stress at both the concentrations with 15 and 16 µmoles/mg of protein/min in Candida viswanathii strain ODBG4, whereas the Cd stress at 0.5mM induced an steady increase with activity of about 16 µmoles and 18 µmoles/mg of protein/min at 48hr and 72hr respectively (Fig. 6 B). The activity of CAT was found to vary based on the metal ion concentration, time and the isolate. The high CAT activity in the basal cells might trigger other relative enzymes such as PO, which might be strategic in mitigating the toxicity of heavy metal ion such as Cd, thus exhibiting steady increase under Cd stress (Pradhan et al., 2017).
3.4.3 Effect of heavy metal-induced oxidative stress on non-enzymatic antioxidant – Reduced GSH
The intracellular GSH plays a key role for maintaining cell homeostasis under normal and stress conditions. In the current study, the levels of reduced GSH in the cells under heavy metal stress was found to be time and concentration-dependent with respect to the treated yeast isolates. In Candida parapsilosis strain ODBG2, the levels of reduced GSH under Cd and Pb stress at 24hr was found to be lesser to that of untreated cells, whereas at 48hr there was an increase by 1.9 fold (Cd 1.0mM) followed by 0.87 fold in Pb (1.0mM) at 48hr. In Candida viswananthii strain ODBG4, a maximum of 1.6 and 1.7 fold increased reduced GSH was found at Cd and Pb (1.0mM) concentration. Increased GSH content at 48hr under Pb stress is similar to the studies by Rehaman and Anjum (2011), with Cd stress in Saccharomyces spp. (Fauchon et al., 2002). Further, decrease in reduced GSH at 72h under metal treatment might be due to the interaction of SH-group of GSH with that of heavy metal, the protective ability towards the toxicity and detoxification of heavy metals (Gharieb and Gadd, 2004; Ilyas et al., 2017) or due to the increased intracellular ions induced activation of other pathways, which leads to imbalance riot in homeostasis (Zhang et al., 2016). The reduced GSH content under different metal stress and at different time intervals is represented in the Fig. 7(A) and Fig. 7(B) for the Candida parapsilosis strain ODBG2 and Candida viswanathii strain ODBG4 respectively.
3.5. Alterations in Secretory proteins upon different heavy metal stress in Candida parapsilosis strain ODBG2 and Candida viswanathii strain ODBG4 by SDS-PAGE.
Candida parapsilosis strain ODBG2 and Candida viswanantii strain ODBG4 extracted at 48hr of post-heavy metal stress (0.5mM concentration) of Pb(II), Cr(IV) and Cd (II) showed similar secretory protein profiles on 10% SDS-PAGE (Fig: 8.A and Fig. 8.B). In the present study, the SDS-PAGE profile showed two (2) over-expressed and intense protein bands in the molecular range of ~ 40-45kDa under Cd stress and faint polymorphic bands in the range of ~ 66-116kDa. The Pb stress also signposted the secretory protein prominently expressed approximately at − 43-44kDa. The similar profiling under Cd and Pb in these isolates might be attributed to similar kind of signal perception, gene activation, protein and 10 and 20 metabolite production at the species level. Since this work is been carried out for the first time, further studies are required for specifying these expressed protein bands at various molecular ranges.