Chromium (VI) bioremediation potential of filamentous fungi isolated from Peruvian tannery industry effluents

Abstract

The tannery is an important trade in various Peruvian regions; however, tannery effluents are a serious local environmental threat due to its highly toxics components and lack of efficient treatment. The untreated effluents produced by tannery factories in Arequipa Rio Seco Industrial Park (PIRS) have formed a lake in the region nearby. In this work, we study the capability of filamentous fungi species found in this effluents lake with potential for chromium (VI) bioremediation. Fourteen species of filamentous fungi were isolated; only two species were identified Penicillium citrinum and Trichoderma viride, and third strain identified as Penicillium sp. The filamentous fungi showed that are fully tolerant to chromium (VI) concentrations up to 100 mg/L. These fungal strains showed significant growth in chromium (VI) concentrations up to 250 mg/L. Tolerant index (TI) analysis revealed that P. citrinum and T. viride began adaptation to chromium (IV) concentrations of 250 and 500 mg/L, after 6 and 12 days, respectively. When exposed to higher Cr (VI) concentrations (1000 mg/L), only T. viride was able to show growth (enhance phase). Interestingly, one of the significant responses from these fungal strains to increasing chromium (VI) concentrations was an increment in secreted laccase enzymes. Our results show tolerance and adaptation to elevated concentrations of chromium (VI) of these fungal strains suggesting their potential as effective agents for bioremediation of tannery effluents.

Introduction

The tannery is an industry that processes food industry primary waste and provides the raw material for a wide array of products, mainly in clothing industry. It is estimated that 1.76 × 10⁹ m² of leather with a US$7 billion worth is produced annually [1]. In Peru, there are more than 90 tannery factories that provide the raw material used in the Peruvian clothing industry. These tannery factories are mainly distributed in Arequipa, Lima, and Trujillo regions [2]. Arequipa is the second populated Peruvian region and third leather producer with more than 30 tannery factories. These factories are small- and medium-size scale and are concentrated in Rio Seco Industrial Park (Parque Industrial de Rio Seco - PIRS) in Cerro Colorado district, 20 min from car travel from Arequipa city downtown.

Vegetable tanning (using hydrolyzable and condensed tannins) and chromium tanning are the main process for raw hide/skin tanning [3]. Peruvian tannery factories use the chromium-based process; however, approximately 20 to 40% of the chromium used goes directly to effluents [4]. Tannery effluents are among primary sources of chromium pollution of land and water bodies [3, 5,]. Tannery effluents from PIRS factories are deficiently treated, and many of these effluents form streams that end in a nearby ravine named Añashuayco. Over the years, continuous tannery discharge formed a lake in this ravine (Fig. 1a, b) and polluted the soil of the surrounding region; also, it is believed that small streams from this lake go to the Chili River, which is used for agricultural irrigation that produces food for local and regional consumption.

Fig. 1.

The lake formed by tannery effluents in Añashuayco ravine (Arequipa-Peru): a panoramic view; b view of the lake’s main feeding stream

The microbial treatment of industrial effluents is one of the most advantageous and cost-effective methods, using mainly bacterial and/or fungal species [3]. Fungal species are excellent candidates for heavy metal bioremediation, presenting a variety of functional groups in their cell wall, which provide great biosorbent capacity. Moreover, they grow naturally in heavily polluted environments, easy to grow high yielding biomass, and the majority is non-pathogenic [5]. In literature, there are reports that fungal species belonging to various groups including Aspergillus, Penicillium, and Trichoderma are chromium tolerance and have great potential for tannery effluents bioremediation [6].

In this work, we investigated the tolerance of three fungal species to a high concentration of chromium (VI) that were isolated from the lake formed by tannery effluents in Añashuayco ravine. This study also reports on the chromium-induced expression of laccase in these fungi species, which suggests their high potential for tannery effluent bioremediation and biotechnological applications.

Material and methods

Sample collection and fungal species isolation

The Añashuayco ravine, in which tannery effluents formed a lake, is located to the northeast (Zone 19K - 221607.99m E –8189112.45m S UTM) from Arequipa city downtown, in Cerro Colorado district (Arequipa-Peru). Samples were collected using sterile bottles of 250 mL capacity and transported at 4 °C. The isolation of fungal strains were carried out following the dilution method in Sabouraud Agar (Merck) described by Nuñez [7]. Axenic cultures were obtained after 7 days of sequential subculture at 21 °C. Taxonomic identifications of isolated strains were carried out by microscopical morphology identification following the method described by Macalupú et al. [8]. The validation of the taxonomic identification of the fungal strains, which showed chromium (VI) tolerance, was made by the Laboratorio de Biotecnología Micológica of Cayetano Heredia University (Lima, Peru) [9].

Experimental design

Pre-tolerance to Cr (VI) studies was carried out in Petri dish plates using Sabouraud Agar supplemented with a unique concentration of K₂Cr₂O₇ (100 mg/L), the isolated strains were incubated for 7 days at 21 °C, and then, the strains that showed most growth were chosen to carried out the tolerance experiments. Chromium (VI) tolerance studies of resistant fungal strains were carried out for 21 days in solid medium Sabouraud Agar supplemented with K₂Cr₂O₇ to final concentrations ranging from 50 to 5000 mg/L. Fungal growth was measured using a digital caliper (Uyustools) following the method described by Valix and Loon [10] from the point of inoculation to the end of the longest hypha.

These measurements were calculated as follows: (1) tolerance index (TI) defined as the ratio between the fungi growth in metal presence and the fungi growth in metal absence during the same period [11]. (2) The minimum inhibitory concentration (MIC) that according to Andrews [12] is define as the lowest concentration of a substance that will inhibit the visible growth of a microorganism.

Studies of the effects of different concentrations of Cr(VI) over biomass production of fungal strains were carried out in liquid medium (100 mL) with the following composition: 10 g/L glucose, 1 g/L NH₄NO₃, 0.8 g/L KH₂PO₄, 0.2 g/L Na₂HPO₄, 0.5 g/L MgSO₄·7H₂O, 2 g/L yeast extract [13]. The medium was buffered with HCl 0.1 M to achieve acidic conditions (pH 5). The medium was supplemented with K₂Cr₂O₇ to final concentrations of 50, 500, 1000, 2500 and 5000 mg/L and the strains were allowed to growth during 5 days at 21 °C under 120 rpm agitation (Orbital agitator Sho 2D, Diahann, South Korea). Fungal biomass was obtained by filtrating the medium with Whatman paper filter No. 40; then, the biomass was dehydrated in a stove at 80 °C. The dried material was weighted using a digital balance RADWAG AS3Y (Radwag Balances and Scales, Poland), and the resulting value was defined as the dry biomass weight. Biomass inhibition percentage was calculated using untreated and treated dry biomass weight as described by Nongmaithem et al. [14]. For laccase activity determination, filtrate obtained previously was centrifuged (Table Top Centrifuge PLC-05, Gemmy Industrial Corp) at 5488×g for 10 min; the supernatant (enzymatic extract) was stored at − 4 °C until use. Laccase activity was measured colorimetrically following the method described by Manjarrés et al. [15] by oxidation of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS; Sigma-Aldrich, St. Louis, USA). Briefly, a quartz cuvette (1 cm path) was mixed 750 μL of buffer acetate 0.1 M pH 5, 1500 μL enzymatic extract, and 250 μL ABTS solution (1 mM) in a final volume of 2500 μL. Absorbance changes were measured at 420 nm in a UV-VIS Pharo 300 spectrophotometer (Merck). Laccase activity was expressed in μmol min⁻¹ L⁻¹ using the ABTS molar coefficient extinction 3600.

Statistical analysis

Results were report as mean ± SD of three replicates. The significance of differences among means was assessed by analysis of variance (ANOVA) followed by Tukey test. Statistical analyses and figures were made using Origin v8.0891 (OriginLab Corporation, USA) software. Significant differences were considered if p < 0.05.

Results and discussion

Samples were collected at three points (P1, P2, and P3) from the lake in Añashuayco ravine. These samples were isolated fourteen strains (Table 1): four strains from point P1, five from point P2 and point P3, each. Three isolated strains (P1-01, P2-01, and P3-01) showed significant growth (diameter > 4 cm) in the pre-tolerance study and were identified as Penicillium citrinum (P2-01), Penicillium sp. (P3-01), and Trichoderma viride (P1-01). Several studies [16–18] show that filamentous fungi from the phylum Ascomycota are common in mycoflora in industrial effluents. The growth capacity of fungal strains at different chromium (VI) concentrations was evaluated in solid medium (Fig. 2a–c). All three strains showed similar growth at 50 mg/L when compared with its control (medium without metal); T. viride showed the fastest growth reaching a maximum after 6 days; on the other hand, both Penicillium species reached maximum growth after 12 days. When P. citrinum and T. viride were exposed to 250 mg/L (Fig. 2a–c, open triangle) of Cr (VI) showed a significant (p < 0.05) retardation in their growth rate when compared to control, reaching a maximum growth after 18 days. However, the growth of both fungal strains was similar to each other under the same Cr (VI) concentration, suggesting a similar tolerance mechanism. These results are interesting since the growth of fungal strains in the presence of a heavy metal usually shows differences in their growth [10, 11, 19]. On the other hand, Penicillium sp. has a stalled growth suggesting reduced tolerance to the same Cr (VI) concentrations (Fig. 2c). Exposing fungal strains to 500 mg/L of Cr (VI) resulted in the maximum growth of T. viride and P. citrinum were reduced by half (Fig. 2a, b, inverted triangle). After 15 days, P. citrinum showed a small increment in its growth, when compared with T. viride, although both strains showed similar growth after 21 days. Dugal and Gangawane [20] reported a Penicillium strain with low tolerance to Cr (VI); accordingly, our results shows Penicillium sp. strain has low tolerance to this metal. Our growth results suggest that fungal strain is one of the factors that influence tolerance responses to heavy metals high concentration. Indeed, De Sotto [21] reported that different strains of Aspergillus and Penicillium showed different tolerance and adaptation capacities to the same heavy metal. Moreover, the authors demonstrate that pH of the medium is the main parameter that influences fungal sorption, which is one of the principal fungal mechanisms of heavy metal tolerance. When fungal strains were exposed to 1000 mg/L of Cr (VI) (Fig. 2a–c, hexagon), only T. viride showed some growth; above this concentration, none of the fungal strains tested showed growth, indicating that 500 mg/L of Cr (VI) is the maximum concentration tolerated by these strains. Heavy metals like chromium, cadmium, and cobalt are toxic for microbes [16, 22]; however, a variety of fungal species thrive in environments with significant concentration of these metals [23, 24]. In this regard, Penicillium and Trichoderma species isolated from heavy metal pollutant effluents from different regions showed similar tolerance [20, 23, 25].

Table 1.

Isolated fungal strains and growth in solid medium containing Cr (VI)

Cr (VI) (mg/L)		Fungi strain
	P1-01	P1-02	P1-03	P1-04	P2-01	P2-02	P2-03	P2-04	P2-05	P3-01	P3-02	P3-03	P3-04	P3-05
0	+	+	+	+	+	+	+	+	+	+	+	+	+	+
100	+	–	–	–	+	–	–	–	–	+	–	–	–	–

⁺Significant growth > 4 cm

⁻No growth < 2 cm

Fig. 2.

a Growth curves of aTrichoderma viride; bPenicillium citrinum, and cPenicillium sp. in presence of different Cr (VI) concentrations. Control: growth medium without Cr (VI). Results are expressed as media ± SD (n = 3). *Significant difference (p < 0.05) compared with control. **High significant difference (p < 0.01) compared with control

The fungal species resistance and adaptation to various heavy metals can be evaluated using the tolerance index (TI). Valix et al. [11] postulate that plotting time vs TI shows a predictable fungal adaptation pattern of five phases: (a) an initial lag phase, (b) a phase of rapid growth followed by (c) retarded growth phase; this continues with a (d) stalled growth phase and finally an (e) enhance growth phase. Our TI results show that the three fungal strains are completely adapted (TI ≥ 1) to 50 mg/L chromium concentration (Fig. 3a). The TI values to 250 mg/L of Cr (VI) (Fig. 3b) of P. citrinum and T. viride show adaptation (TI ≥ 1) after 18 days. Both strains showed a similar pattern of reduced stalled growth phase and a prolonged enhance growth phase. Penicillium sp. strain (Fig. 3b, triangle) shows a retarded and stalled growth phases and is extended with a discrete enhance growth phase, which confirms that this fungal strain is highly sensitive to relatively elevated Cr (VI) concentrations. When challenged with 500 mg/L Cr (VI) concentration, P. citrinum and T. viride showed similar adaptation patterns, with the five phases clearly distinguish (Fig. 3c); lag, rapid growth, retarded growth, and stall growth phases develop in 12 days. We notice an extended enhance growth phase suggesting a slow but continuous adaptation process of these fungal strains. However, after 21 days, TI values for both strains reached only an average value of 0.5. These results suggest that adaptation of both strains to this Cr (VI) concentration may need more time. Interestingly, other reports [10, 11, 19] report that Penicillium strains needed shorter periods (< 12 days) of adaptation to toxic metals like Ni or Co. However, Cr (VI) (chromate and dichromate) is soluble, very toxic, and mutagenic; moreover, due to its chemical structural similarity with sulfate, it can cross over the cellular membrane via the sulfate uptake pathway [26]. Thus, the high toxicity of Cr (VI) may be one of the reasons that P. citrinum and T. viride need more time adapting to this metal.

Fig. 3.

Tolerance behavior of Trichoderma viride (open square), Penicillium citrinum (open circle), and Penicillium sp. (open triangle) to chromium (VI) concentrations: a 50 mg/L; b 250 mg/L; c 500 mg/L; d 1000 mg/L. Results are expressed as media ± SD (n = 3). *Significant difference (p < 0.05) among treatments

Similar to fungal growth results, TI values for both Penicillium strains confirm that they were not able to adapt to chromium (VI) concentrations above 1000 mg/L (Fig. 3d). On the other hand, TI results also confirm the marginal tolerance of Trichoderma viride to high Cr (VI) concentrations. Overall, T. viride showed a high chromium MIC = 1000 mg/L; on the other hand, Penicillium strains were sensible to chromium with a MIC = 500 mg/L. Fungi strains show great tolerance and adaptability to high concentrations of heavy metal present in the environment. Moreover, the tolerance and adaptation of fungal strains can be continuously enhanced by selecting fungal strain in the enhance phase (TI ≈ 1) and exposing them to a higher metal concentration [10, 11, 19], which is a great quality for developing bioremediation methods based in these microorganisms. Our results of fungal growth and tolerance of Penicillium citrinum and Trichoderma viride in the presence of different concentrations Cr (VI) suggest that both strains can be submitted to adaptation experiments. These adaptation experiments can be initiated by exposing the P. citrinum and T. viride strains in the enhanced phase (250 mg/L of Cr (VI)) to an initial Cr (VI) concentration of 300 mg/L. Moreover, extending the time of exposure to chromium 500 mg/L can be considered a second strategy to obtain adapted strains; both approaches can improve the bioremediation application prospects of these two fungal species.

So far, biosorption and bioaccumulation are the overall process by which microorganism tolerate toxic levels of heavy metals in the environment. However, as reported by Das and Guha [27], both process function in different times and through different molecular mechanisms. Indeed, in a living cell, initial uptake of Cr (VI) occurs in the surface of the cell wall. These processes are based mainly in the reduction of extracellular chromate by anionic absorption, reduction by adjacent electron donor, and release by electronic repulsion [28, 29]. These mechanisms are depended of the biomass produced by the fungal strain, which gives the sufficient cell wall surface area to expose enough functional groups. The growth of Penicillium citrinum and Trichoderma viride provides enough initial biomass when expose to 50 mg/L of Cr (VI). However, when exposed to 250 and 500 mg/L of Cr (VI), the biomass produced by these fungal strains was overwhelmed by the metal concentration, resulting in a reduced (biomass) and short (time) rapid growth phase. The bioaccumulation process described above is metabolism independent; however, there is also bioaccumulation processes that are metabolism dependent aiming to produce reducing power to reduce Cr (VI) to Cr (III) [28]. Aspergillus sp., Penicillium sp., and Trichoderma inhamatum reduce chromium using the reducing power generated by carbon metabolism [30]. Other mechanism permits chromate accumulation in the cytoplasm through the sulfate transport system [27]. From our results, it is evident that P. citrinum and T. viride rely heavily on a bioaccumulation process, especially when exposed to Cr (VI) concentrations equal or higher to 250 mg/L. Currently, our laboratory is conducting adaptation experiments of both fungal strains to 500 mg/L of Cr (VI); one of the main objectives of this work is to investigate which mechanism is used by these strains to tolerate and adapt to high chromium concentrations.

Fungi dry biomass determination (Table 2) corroborated the findings by tolerance index, at chromium concentration of 50 mg/L both Penicillium citrinum and Trichoderma viride showed a similar increment in biomass when compared with the growth of the strains in the absence of chromium. In contrast, with a concentration of 500 mg/L, a significant biomass reduction was observed. As stated earlier, chromium is an eminently toxic heavy metal for fungi, moreover, was reported that chromium concentrations can reduce biomass production in some species of Penicillium and Trichoderma [23, 31]. Additionally, fungi strain tolerance and adaptation to heavy metals are affected by various external factors (pH, temperature, etc.) and vary even within the same strain [25], as showed by our results. Thus, these results suggest that both isolated strains possess a remarkable tolerance and adaptation capability to chromium, which promises good results in further adaptation experiments.

Table 2.

Biomass production and laccase activity of Penicillium citrinum and Trichoderma viride at different Cr (VI) concentrations

Strain	Chromium (VI)
	0 (mg/L)		50 (mg/L)		500 (mg/L)		1000 (mg/L)
	Biomass	Laccase	Biomass	Laccase	Biomass	Laccase	Biomass	Laccase
Penicillium citrinum	0.129 ± 0.014	2.716 ± 0.592	0.156 ± 0.012	4.838 ± 0.282	0.013 ± 0.02	35.208 ± 3.171	0.009 ± 0.004	n.d.
Trichoderma viride	0.113 ± 0.004	2.916 ± 0.781	0.13 ± 0.012	3.179 ± 0.282	0.015 ± 0.004	30.679 ± 3.171	0.009 ± 0.002	n.d

Biomass: expressed in milligram of dry weight. Laccase activity: expressed as μmol min⁻¹ L⁻¹. Results are expressed as media ± SD (n = 3). n.d. not determinated

A surprising finding was expression of laccase at high chromium concentrations (500 mg/L) in both Penicillium citrinum and Trichoderma viride (Table 2). The presence of organic pollutants and micromolar concentrations of metallic ion (iron, copper, and manganese) induces expression of laccase enzymes in fungal species; these enzymes are essential to microbial physiology [32, 33]. However, there are reports showing enhance laccase expression in fungal species as a response to 100 mg/L of cadmium, incrementing laccase activity by 19-fold and this was related to biomass increment [34]. In comparison, P. citrinum and T. viride showed 10-fold increment of laccase activity, with a reduction of fungal biomass. It appears that both strains increment enzyme expression at the expense of biomass increment. Fungal laccases exert several physiological roles and possess a high activity on organic pollutants that are present in tannery effluents, which is suggested that the increasing expression of this enzyme in fungal strains is a response to the presence of these compounds [35, 36, 37]. Additionally, it is desirable that a microbe showed bioremediation capabilities of recalcitrant organic pollutants, since these pollutants are resistant to a variety of treatments [38]. So far, our results suggest that laccase expression is one of the principal responses of P. citrinum and T. viride to the conditions found in the lake formed by tannery effluents in Añashuayco ravine, and currently, our laboratory is addressing this issue.

In conclusion, from a lake formed by tannery effluents were isolate two fungal species identified as Penicillium citrinum and Trichoderma viride, which showed tolerance to Cr (VI) concentration up to 250 mg/L. These fungal species showed be able to adapt to 500 mg/L of Cr (VI), and one of the principal adaptations of these fungal strains to Cr (VI) stress is laccase enhance expression. These results show that both fungal species have a great potential for tannery effluents bioremediation, which are a great health and environmental problem in Arequipa region.

Funding information

This study was financed by the UNSA-INVESTIGA fund contract number IBA-0030-2017 and was part of the undergraduate work of Shirley Vanessa Zapana-Huarache.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.