- Email: [email protected]
An eco-friendly approach for minimizing pollution of metal from municipal solid waste leachate in India
Accepted Manuscript An eco-friendly approach for minimizing pollution of metal from municipal solid waste leachate in India Abhishek Kumar Awasthi, Akhilesh Kumar Pandey, Jamaluddin Khan PII:
S0959-6526(16)31529-3
DOI:
10.1016/j.jclepro.2016.09.188
Reference:
JCLP 8148
To appear in:
Journal of Cleaner Production
Received Date: 16 June 2016 Revised Date:
11 September 2016
Accepted Date: 23 September 2016
Please cite this article as: Awasthi AK, Pandey AK, Khan J, An eco-friendly approach for minimizing pollution of metal from municipal solid waste leachate in India, Journal of Cleaner Production (2016), doi: 10.1016/j.jclepro.2016.09.188. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
An eco-friendly approach for minimizing pollution of metal from municipal solid waste leachate in India Abhishek Kumar Awasthi a, c * 1, Akhilesh Kumar Pandey a, b, Jamaluddin Khan a a
RI PT
Mycological Research Laboratory, Department of Biological Sciences, Rani Durgavati University, Jabalpur (M.P.), India. b Madhya Pradesh Private Universities Regulatory Commission, Bhopal, India c Department of Microbiology and Biotechnology, V.A.B. College, Chhatarpur, M.P. India 1 Present address: School of Environment, Tsinghua University, Beijing P.R. China 100084
SC
*Corresponding author: A. K. Awasthi, Department of Microbiology, V.A.B. College, Chhatarpur, M.P. India. (M.P.), India. E-mail- [email protected]
Abstract
M AN U
Municipal solid waste (MSW) leachate management has become an important issue worldwide. The present study evaluated the potential of indigenous fungi for biosorption of Cd2+ from leachate. Three isolates—Trichoderma harzianum, Aspergillus niger and Aspergillus flavus were selected based on their ability to exclude the Cd2+ at the preliminary stage. Among these, Trichoderma harzianum was found to be an excellent fungus for cadmium absorption. The highest biosorption rate for Cd2+ was achieved under the conditions: temperature 450C (64.87%), pH 6.0 (64.78%) and
TE D
spore concentration [105cfu ml−1] (64.41%). The obtained data indicated that the developed fungal consortium is very efficient and effective for cadmium removal (72.41%), thereby demonstrating that it is a promising solution for removing toxic metals from MSW leachate.
AC C
1. Introduction
EP
Keywords: Biosorption, Cadmium metal, Indigenous fungi, Municipal solid waste; Waste management.
Municipal solid waste has become the fastest-growing waste stream and its proper collection, recycling and final disposal is one of the most challenging and problematic tasks throughout the world (Yang et al., 2015). The World Bank report estimated that the generation rate of MSW was 0.34 kg/capita/day and 109,589 tonnes/day in 2005. In addition, these figures were expected to rise to 0.7 kg/capita/day and 376,639 tonnes/day by 2025 (Hoornweg and Tata, 2012). The MSW is commonly managed with methods, such as composting, landfill disposal, incineration and open dumping. Among the current management practices, the cheapest—open disposal—is the most common, particularly in developing countries, such as India. However, such
ACCEPTED MANUSCRIPT
practices are a potential threat to the surrounding environment. Rainfall percolating through these open dumping sites triggers biological processes, chemical reactions and physical changes as the MSW degrades, generating a highly contaminated liquid known as leachate. Leachate contains high concentrations of dissolved organic materials, inorganic substances and two categories of heavy
RI PT
metals: essential metals (copper, manganese, zinc and iron), and non-essential metals (cadmium, lead, mercury and nickel). These metals are among the major environmental pollutants because of their high toxicity. However, the exact composition of leachate can vary and depending on the type of waste, climatic conditions, rainfall patterns, hydrological factors and age of the disposal waste
SC
(Ghosh et al., 2015). Numerous physico-chemical methods are commonly employed to remove heavy metals from effluent. These physical and chemical treatment processes are not effective for
M AN U
the management of MSW leachate. In fact, the less ecofriendly technologies are often inefficient, highly expensive and also possess secondary pollution (Fomina and Gadd 2016). Hence, the development of new sustainable and ecofriendly technologies is essential for the treatment of leachate.
Over the past few years, microbial treatment approaches have been attracting more attention, owing to their efficiency, comparative cost-effectiveness and eco-friendliness (Nongmaithem et al.,
TE D
2016; Zhao et al., 2016; Vijayaraghavan and Balasubhaniam 2015). These methods have many advantages, such as easy handling and maintenance (requiring less technical support), higher metal uptake capacity with high treatment rates, minimal sludge production, and high reusability capabilities (Bazrafshan et al., 2016; Fomina and Gadd 2014; Lo et al., 2014). The mechanism of
EP
removing cadmium through microorganisms is a highly complex process because there is competition among various substances, for surface binding sites. The metal biosorption mechanism
AC C
depends on chelation, ion exchange, complexion, adsorption, absorption or micro-precipitation or a combination of these methods (Pandey et al., 2013). The transfer of a metal from outside a cellular membrane into the intracellular space is a metabolism-dependent process governed by a living microbial system. This process is also a microbe’s active defense system, which is potentially capable of tolerating metals. Several microorganisms, including the fungi Pencillium, Rhizopus, Aspergillus and Mucor, as well as yeast and various bacteria, have been used in metal-polluted sites, owing to their continuous enrichment capabilities and highly adaptive nature (Vijayaraghavan and Balasubramanian, 2015; Pandey et al., 2013).
ACCEPTED MANUSCRIPT
Fungi have a bulky biomass and many strains are capable of accumulating either a single heavy metal or multiple metals simultaneously (Mishra and Malik, 2014). The heavy metals removal depends on several factors, such as pH, temperature, incubation period, and inoculum concentration (Koelmel et al., 2016; Chojnacka K., 2010; Sun et al., 2010). The protonation and deprotonation of
RI PT
functional and carboxyl groups play a significant role in the process of metal biosorption (Lo et al., 2014). Mosbah and Sahmoune (2013) stated that, the operational pH range could affect the accessibility of sorption sites on the cell wall surface and the capability of sorption sites to bind metal ions. Thus, these microbes could hypothetically be applied in situ in leachate treatment. Actual
SC
utilization of these strains would require that they have extraordinary tolerance to Cd2+: able to efficiently eliminate it. Yet, in spite of numerous studies on the ability of potential fungi isolates to
M AN U
extract Cd2+, the reaction of fungal strains to this metal has not been thoroughly studied. The objective of this investigation was to assess the practicability of application indigenous fungus from MSW site and development fungal consortia (Trichoderma harzianum, Aspergillus niger and Aspergillus flavus) for the removal of cadmium metal from MSW leachate. The removal efficiency of cadmium metal under diverse conditions (pH, temperature, inoculum concentration and
TE D
incubation period) was examined.
2. Materials and methods
2.1. Sample collection, processing and analysis
The MSW leachate samples were collected from different dumping site around Jabalpur city,
EP
Madhya Pradesh (M.P.) India. During the survey-it was observed that the MSW at site include organic and inorganic matter as well as some of electronic components. The collected leachate
AC C
sample was filtered by Whatman filter paper (Grade 42), before using in the subsequent biosorption experiments or further investigation. The pre-treated and post-treated leachate were also analyzed by Inductive Coupled Plasma Mass Spectrophotometer (ICP-MS) as per Pandey et al. (2013).
2.2. Isolation and identification of indigenous fungal strains
The indigenous fungal isolates were cultivated on potato dextrose agar (PDA) medium through serial dilution method as described by Gautam et al. (2012). The prepared different dilution of leachate sample was inoculated into molten agar medium by using pour plate method under aseptic condition, then the plates were incubated initially for seven days (d) at 28±10C. After desired
ACCEPTED MANUSCRIPT
incubation period, the fungal species were further isolated by single spore culture method (Fig. 1). These isolates were identified on the basis of morphological characteristic, color and appearance of colony and texture, microscopic characteristics (septation of mycelium, shape, diameter and texture of conidia) by slide culture technique and also referred the available manual of principles and
RI PT
practice, (Barnett and Hunter, 1998). These fungal isolates were deposited in the Fungal Germplasm Culture Collection Center (FGCC), M.P. Council of Science and Technology, Jabalpur and also obtained accession number (SI).
SC
Fig. 1. Proposed systematic mechanism for biosorption of cadmium heavy metal employing
M AN U
optimized developed fungal consortium.
2.3. Optimization of environmental condition for fungal growth
The effects of different environmental factors (eg., pH, temperature, inoculum concentration) on the biosorption efficiency of fungal strains in the medium were studied, and control experiments also observed simultaneously. The influence of initial pH on biosorption ability was conducted at a pH range of 3.0 to 8.0, which was adjusted using 0.1 M NaOH and 0.1 M HCl.
TE D
All experiments were carried out in a 250 mL sterilized Erlenmeyer flask containing 150 mL leachate in triplicate. The influence of initial pH on biosorption ability was conducted at a pH range of 3.0 to 8.0, which was adjusted using 0.1 M NaOH and 0.1 M HCl. Batch experiments were performed at different temperature range 28 0C±1 to 60 0C±1 (28 0C, 30 0C, 35 0C, 40 0C, 45 0C, 50 C, 55 0C, 60 0C) with respect to diverse incubation period e.g., 7d, 14d, 21d, 28d, 35d. The
EP
0
cadmium metal concentration after biosorption in leachate were analyzed by using Inductive Coupled Plasma Mass Spectrophotometer (ICP-MS). In the same time, leachate sample does not
AC C
contain fungal spores served as control also studies. The data obtained from the independent experiments were analyzed by used SPSS 15 software. The biosorption potential i.e., the amount of cadmium metal ion (mg/g-1) was calculated by using following equation (1):
Where, ܳ- mg of metal ion biosorbed per g ݅ܥ- Initial metal ions concentration (mg/l) ݂ܥ- Final metal ion concentration (mg/1)
Q =
[Ci − Cf ]v ………………………………………….(1) s
ACCEPTED MANUSCRIPT
ݏ- Wet biomass ݒ- Volume of reaction mixture
3. Results and discussion
RI PT
3.1 Isolation and identification of heavy-metal-tolerant fungi Thirty indigenous fungal isolates were isolated from leachate samples of a municipal solid waste dumping site using standard techniques (Gautam et al., 2012). The effects of cadmium concentration on the growth and morphology of the fungi were also evaluated in a broth culture. Various isolated
supplementary information (SI).
M AN U
3.2 Screening of fungal isolates for tolerance to Cd2+ metal
SC
fungi were identified based on morphological characteristics and these are presented in the
The observed fungal growth of the all the fungal strains, relative to each other, on solid PDA media supplemented with different Cd2+ concentrations. The indigenous fungal isolates showed an ability to grow very rapidly and to cover the entire surface of a petriplate containing PDA Cd2+ concentration levels of less than 0.5 mg/L within a 10-day incubation period, while the remaining strain exhibited very slow growth at a concentration level of 1 mg/L (data not show). At the
TE D
concentration level of more than 1.5 mg/L, three strains—Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus (FGCC#A03)—showed very limited growth. However, no development occurred at all for the other strains, and even for these three strains, the growth rate declined with the addition of extra Cd2+. Nevertheless, the growth of these indigenous
EP
fungi indicated that these strains had potential biosorption ability for Cd2+, and could be used for further experiments.
AC C
These indigenous fungal strains—Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus (FGCC#A03)—showed higher Cd2+-metal tolerance than other identified fungi. Similarly, Pandey et al. (2013) found that indigenous fungal isolates had impressive abilities to remove metal from MSW leachate. The indigenous fungal isolates have more tolerance potential (Pandey et al., 2013). Actually, the removal of heavy metals by microbes seems to depend on both the metal and the fungal species. Among the indigenous strains, we found the two isolates Aspergillus and Trichoderma sp. to be highly Cd2+-tolerant and able to survive in cultures with more than 1.5 mg/L. Our outcomes revealed, that the biosorption ability decreased with higher Cd2+ concentration. In the same time- also noticed that a rise above the initial metal level resulted
ACCEPTED MANUSCRIPT
decrease in the metal exclusion ability of the fungi. It meant that the heavy metals may be harmful to cells when substantial adsorption is established on cell walls. In living fungi, the leading source of metal accumulation is through intracellular uptake via the cell membrane. An efflux tool could also function at a specific metal concentration to prevent further
RI PT
accumulation. The remarkable metal tolerance of fungi is directly related to fungal survival and growth in metal-polluted leachate, and our potential fungal isolates showed considerable metal resistance potential. While, this study did not examine the mechanism of tolerance, these outcomes explained the possible applicability of employing indigenous fungal isolates for cadmium exclusion:
SC
a particularly significant finding for any sites with very high levels of Cd2+ pollution. Similar findings have been reported by many authors. Fazli et al. (2015) suggested that A. versicolor could
M AN U
accumulate Cd2+ at the concentration of 7 mg/g mycelium, Paecilomyces sp. at 5.878 mg/g, Microsporum at 5.07 mg/g, and Trichoderma sp. at 4.55 mg/g. A number of other authors have also studied fungal tolerance to heavy metals (Carrillo and Gonzalez et al., 2012).
3.3 Optimum biosorption conditions of fungal strains
Although numerous microbes have the ability to bind with heavy metals, only a few of them have
TE D
shown excellent metal exclusion through the binding process. Yet maximum metal removal rates can be achieved when optimize the environmental conditions. Such optimal conditions can be established through biosorption experiments. In this study, the optimal conditions for Cd2+ metal exclusion (pH, temperature and incubation period) were determined for highly tolerant isolates of
AC C
(FGCC#A03).
EP
Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus
3.3.1 Effect of pH on metal biosorption Heavy metal biosorption potential can be affected by pH because the acidic level of the metal solution affects the surface binding charge of the fungal biosorbent, and also influences the solubility of the metal. As shown in the Fig. 2, a pH value above 6.0 caused the precipitation of Cd2+. These findings suggested that the Cd2+ biosorption by Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus (FGCC#A03) increased steadily as pH rose, reaching its maximum at pH 6.0 and then declining as the pH fell to 8.0. This reduction at pH>6 might be caused by complexation with soluble organic ligands (Gola et al., 2016). Low pH (pH<4) also decreased the
ACCEPTED MANUSCRIPT
biosorption rate, with lower pH values corresponding to lower biosorption rates (Fan et al., 2014). This pattern occurred in all three of the fungi strains—Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01) and Aspergillus flavus (FGCC#A03). The biosorption of metal is highly correlated with the pH of the solution is that it can impact the
RI PT
metals’ chemistry and the activity of functional groups (carboxylate, phosphate, and amino groups) on the cell wall, because of competition between metal ions and binding sites (Bazrafshan et al., 2016; Nongmaithem et al., 2016). Studies conducted by Fan et al. (2014) suggested that the biosorption ability of metal is lower at lower pH levels because of hydrogen ions’ competition with
SC
metal ions present on the fungal cell wall sites. However, metal precipitates at higher pH levels, inhibiting the contact of metal with most fungal biomass (Razarinah et al., 2014)—a phenomenon
M AN U
that accounts for the drop in biosorption rate as pH increases above 6.0.
Fig. 2. Effect of pH on biosorption of cadmium metal employing developed fungal consortium.
3.3.2. Effect of temperature on metal biosorption
Temperature is another important factor in the biosorption process. As shown in the Fig. 3, the
TE D
biosorption of Cd2+ removal increased from 33.2% to 63.98% as the temperature rose from 28 0C to 45 0C, and then abruptly decreased between 45 0C and 60 0C, for all the inoculum concentrations of the three individual fungal strains [Trichoderma harzianum (FGCC#A29), Aspergillus flavus (FGCC#A01), and Aspergillus niger (FGCC#A03)], although the maximum Cd2+ biosorption was
EP
51.01% at 50 0C for the Trichoderma harzianum (FGCC#A29). Hence our investigation confirmed that temperature plays a significant role in the biosorption process (Lo et al., 2014; Ahalya et al.,
AC C
2003). Pandey et al. (2013) suggested that maximum metal uptake activity is correlated with higher temperature because high temperature increases the affinity of metal-binding sites on fungal cell walls. Nevertheless, room temperature is suitable—although not optimal—for an effective biosorption process. This correlation between biosorption ability and higher temperature needs further, more detailed investigation. Fig. 3. Effect of temperature (0C) on biosorption of cadmium metal employing developed fungal consortium.
ACCEPTED MANUSCRIPT
3.3.3. Effect of inoculum concentration on metal biosorption The most frequently isolated indigenous fungal isolates from the leachate sample were, in order: Aspergillus sp., Alternaria sp., Curvularia sp., Fusarium sp., Mucor sp., Rhizopus sp. Of these indigenous isolates only three fungal strains were selected based on their potential capacity for metal
RI PT
biosorption at various inoculum concentrations. The fungal isolates Cladosporium oxysporum (FGCC±A10), Chaetomium globosum (FGCC#A11) and Cunningmella echinulate (FGCC#A12) showed relatively low levels of cadmium biosorption compared to the other isolates. The fungal isolates showing the highest cadmium metal tolerances through ionic association among
SC
extracellular polysaccharides, proteins and chitins, were in order: Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01) and Aspergillus flavus (FGCC#A03). Other fungi,
M AN U
such as Ganoderma austral, Aspergillus terreus, A. lentulus and Rhizopus oryzae have been reported on by researchers studying MSW leachate biosorption (Mishra and Malik, 2014; Razarinah et al., 2014). Furthermore, during the standardization process, it was noted that maximum biosorption of Cd2+ metal was accomplished using Trichoderma harzianum FGCC#A29 (51.01%) followed by Aspergillus niger FGCC#A01, (40.62%) and A. flavus FGCC#A03 (37.65%) and that biosorption activity can be affected by, inoculum concentration (Fig. 4), temperature pH and incubation time
TE D
(Fig 5).
cadmium metal
EP
Fig. 4. Effect of inoculum concentration (cfu ml−1) of developed fungal consortium on biosorption of
Fig. 5. Effect of incubation period on biosorption of cadmium metal employing potential fungal
AC C
strains.
3.3.2. Development of fungal consortium It was observed that excessive cadmium concentration was able to inhibit fungal growth during the lag phases. A single fungal strain, cannot effectively treat leachate. It must be tested in a synergistic relationship with other potential fungal isolates, and then a fungal consortium can be developed based on their synergistism performance. In this study, it was found that the developed consortium performed well for the biosorption of Cd2+ (72.41% and 30.34% by fungal consortia and natural flora respectively) from MSW leachate at pH 6.0±1; 450C; 35d), as shown in the fig. 6.
ACCEPTED MANUSCRIPT
Hence, it can be assumed that a standard combination of these three fungal isolates would be helpful in developing an effective leachate treatment (Fig. 5). Therefore, this studied indicates synergistic relationship in order to better understand the developed fungal consortium. Bazrafshan et al. (2016) stated that metal biosorption directly related to valency and atomic number, and that Cd2+ had a
RI PT
greater affinity to the binding sites of our developed fungal consortium than to any of the individual fungal strains. The biosorption potential percentage of Cd2+ using the developed consortium was higher than when using the individual fungal isolates, under all growth conditions. Nevertheless, an optimal fungal growth environment was an essential factor for increasing the percentage of metal
SC
biosorption.
The efficacy of indigenous fungi in the management of heavy-metal-contaminated MSW
M AN U
leachate is a promising approach because the mechanism involves both extracellular adsorption and intracellular accumulation or absorption: in other words, biosorption. This promising strategy— using fungi for the biosorption of Cd2+ metal—has been previously reported (Bazrafshan et al., 2016; Zhu et al., 2016; Silva et al., 2014;). In order to achieve these results, a fungus must have a very high tolerance level to heavy metals. Many fungal strains were tested and failed to propagate because of the toxicity of the heavy metals, making them unsuitable. Zhang et al. (2016) stated that
TE D
acclimatization is one possible way to enhance a microorganism’s resistance to heavy metals; they used a wild strain of Pleurotus ostreatus HAU-2, inoculated with media containing different concentrations of metal (Pb), and found that after 90 days of incubation, the fungus’s ability to survive the metal’s toxicity was enhanced significantly. This result indicates that using indigenous
isolates.
EP
fungi for metal removal from leachate could be more efficient than using non-indigenous fungal
AC C
Many studies have shown that heavy metal biosorption employing fungi have great ability because their cell walls are composed of proteins, lipids, and polysaccharides—different functional groups that are able to bind to metal (Vijayaraghavan and Balasubramanian, 2015). A cell wall made of chitin and chitosan (mixed polysaccharides) plays an important role in metal binding. Hence fungi, such as Aspergillus niger can remove metals with its spores and can therefore act as an excellent biosorbent for metals. Cd2+ metal biosorption employing several different fungi, at various concentration levels, has been well documented by several researchers (Kapoor and Viraraghavan, 1998). Exact results have varied, however, among different investigations, owing to the type of microbes used, the inoculum concentration, temperature, and pH as well as the experimental
ACCEPTED MANUSCRIPT
methods. For instance, Xu et al. (2015) studied cadmium detoxification using Penicillium chrysogenum XJ-1 with a method that included biosorption, cellular sequestration, and antioxidant defense, and their findings suggested that Cd2+ metal had been effectively eliminated from contaminated soils (at the concentration 5–50mg kg−1). Still, this fungal strain needs more study,
RI PT
possibly by mixing it with compost or biochar for improving the plant growth and development. Exposure to cadmium can cause modifications to fungal morphology, in all the indigenous fungal strains. Some researchers have suggested that it forming colorful mycelia due to admits of heavy metals on media (Shakya et al., 2015). Vijayaraghavan and Balasubhaniam (2015)
SC
emphasized that most of the studies carried out previously had certain constraints for successful metal removal, such as only working with synthetic solutions.
M AN U
Fig. 6. Comparative evaluation of biosorption potential by developed fungal consortium and natural flora.
Many researchers have carried out detailed study on Cd2+ biosorption, using various fungal strains, such as Aspergillus & Rhizopus [2.72-2.91 mg/g-1 & 2.72 mg/g-1] (Zafar et al., 2007) and Aspergillus niger [1.31 mg/g-1] (Kapoor and Viraraghavan, 1998). One of their findings was that excessive concentration levels of cadmium metal could cause discoloration of the fungus, such as
TE D
changing pink to white, as in the case of Paecilomyces sp. and Paecilomyces sp. genera. Researchers have also reported pigment production in the fungal cell and in cell-free media, together with metal ion precipitation on cell walls. This production may act as a preliminary defense, to protect the
EP
protoplast from metal toxicity (Fazli et al., 2015). Microbes sometimes produce metal-chelating proteins, such as metallothionein and glutathione, which are essential for the reduction of cadmium toxicity. After saturating the binding sites of the
AC C
fungal cell walls, the metal crosses into the vacuoles, and is then chelated with different types of organic acid (Shakya et al., 2015). The protonation and deprotonation of functional groups also make significant contributions to the biosorption process, because pH may alter the approachability of sorption sites for binding metal ions to a specific surface site. Although higher metal removal could be accomplished at either a neutral or an alkaline range of pH, that reflects the deprotonation of carboxyl group (Pandey et al., 2013). Some other researchers have also determined that different metal concentrations can affect fungal growth rates (Aspergillus carbonarius, Fusarium sp. and Penicillium). It may be that the metals affect their enzymatic activity, resulting in a decrease in the
ACCEPTED MANUSCRIPT
production of conidiophores (Xu et al., 2015). Carrillo and Gonzalez (2012) found that the indigenous fungus Scleroderma citrinum proved to have a good tolerance level for Cd2+ (78%-95%) and that this tolerance depended not only on the metal concentration but also on whether the strain is indigenous.
RI PT
Some authors have proposed that the main mechanism of biosorption of metal is related to ion exchange (Ahalya et al., 2003). Microbes vary in their compositions of membrane lipid cell walls, and different growth phases of cell membranes may directly affect their susceptibilities to toxic substances (Fan et al., 2014). In other words, the fungal cell wall significantly contributes to the
SC
adsorption of Cd metal ions, and the–OH and–C=O groups on their cell surface are responsible for the Cd2+ binding. The cell wall is the first line of defense and also protects the protoplast from metal
M AN U
poisoning for short periods of metal threat. When the saturation stage is reached, most intracellular metals have a tendency to be transported to vacuoles and then chelated with different organic acids: e.g., citric, oxalic, etc. to accomplish metal compartmentalization. Toxic metal deposition on cell walls and vacuolar compartmentalization of microbes are well known to be metal detoxification tools. Thus, intracellular compartmentalization could play a key role in the decontamination and tolerance of Cd2+ in the fungal strains.
TE D
Salunkhe et al. (2015) suggested that morphology, such as fibrousness, hyphae, pelletization, and clumping of filamentous fungi play crucial roles in the uptake of metabolites and enzyme production. Zhu et al. (2016) stated that living cells always have better capabilities as a biosorbent for metal (Ni2+, Cd2+, Cu
2+
and Cr
3+
), than non-living substances, because the available binding
EP
sites are very limited on non-living absorbent, and excess metal ions could neither bind on it nor be removed from the solution. The biosorption capacity of living cells, however, might be improved
AC C
with the application of more energy, so that they could deal with increases in the metal concentration.
The main benefits of biosorption are that it is cost effective, non-invasive, sustainable, and more ecofriendly than conventional technologies, such as physical, chemical, incineration etc. Furthermore, this process can be made site-specific, reducing health risk, and it permanently detoxifies the waste, eliminating long-term liability. It could also be combined with physical or chemical technologies. The process can be varied by using different types of biosorbents, such as microbial biomass (i.e., bacteria, fungi and yeast), algal biomass, proteins, and other biomaterials.
ACCEPTED MANUSCRIPT
Furthermore, the strains could be used as successful microbial agents for solving the problem of cadmium metal pollution in the natural environment. As shown in the above-discussed studies, the developed fungal consortium is promising candidate for the detoxification of heavy metal from MSW leachate. Yet despite its many
RI PT
advantages, many challenges still remain for biosorption. For example, Vijayaraghvan and Balasubramanium (2015) have suggested that significant work is still needed to improve biosorption efficiency. The dearth of operational environments and the need for optimization could restrict the practical and effective application of developed consortia in the transformation process. Chojnacka
SC
(2010) believed that, this method is needed to supplement the external source of energy (eg. Sucrose) to growing cells. Though, if the suitable fungal strains are identified, it is promising to
M AN U
recommend a self-replenishing method whereby microbial cells which absorb or adsorb the contaminants (either organic compounds or inorganic ions). Although, this approach has not been explored systematically, yet. Because there might be a demand for processing vast amounts of heavy metals having a toxicological impact on the surrounding environment. Gadd (2009) said that, the limitations such as, shorter life time of biosorbents compared to conventional approach. Large-scale implementation of biosorption is not currently practical, and further study is necessary, to deal with
TE D
heavy-metal contamination of the environment.
The next step is to determine exactly how the growth and development of these fungal isolates can be extended to remediate the cadmium metal-polluted soils of the Jabalpur MSW site. More
4. Conclusion
EP
advanced research is warranted, to find more eco-friendly disposal methods.
This research provided an opportunity to evaluate the biosorption potential of indigenous fungal
AC C
isolates. Results clearly revealed that inoculation of potential strains in leachate can effectively and efficiently accelerate the biosorption mechanism and remove cadmium metal, while natural flora showed weak performance. The optimal conditions were: spore concentration 10-5, pH 6.0±1 and temperature 450C. These conditions achieved significant biosorption (72.41%) for cadmium metal. This method is an ecofriendly, cost-effective and energy-saving approach to leachate treatment. More research is needed, however, before implementing the technology on a large scale.
Acknowledgements
ACCEPTED MANUSCRIPT
The authors are thankful Head, Department of Biological Science, R.D. University, Jabalpur for providing laboratory facilities and also thankful to Municipal Corporation Jabalpur (M.P.) India for their support.
RI PT
Appendix A. Supplementary information (SI) attached in this manuscript. References
Ahalya, N., Ramachandra T.V., Kanamadi R.D., 2003. Biosorption of Heavy Metals. Res. J. Chem. Environ. 7(4), 71-78.
Barnett, H.L., Hunter B.B., 1998. Illustrated genera of imperfect fungi (3rd Edn.) ABS Press. The Am. Phyto.
SC
Soc., Minnesota.
Bazrafshan, E., Zarei A.A., Mostafapour F.K., 2016. Biosorption of cadmium from aqueous solutions by Trichoderma fungus: kinetic, thermodynamic and equilibrium study. Desalination Water Treat. 57
M AN U
(31), 14598-14608.
Carrillo, G.R., Gonzalez-Chavez Mdel, C., 2012. Tolerance to and accumulation of cadmium by the mycelium of the fungi Scleroderma citrinum and Pisolithus tinctorius. Biol. Trace. Elem. Res. 146, 388-395.
Chojnacka, K., 2010. Biosorption and bioaccumulation e the prospects for practical applications. Environ. Inter. 36, 299-307.
TE D
Fan, J., Okyay, T.O., Rodrigues, D.F., 2014. The synergism of temperature, pH and growth phases on heavy metal biosorption by two environmental isolates. J. Hazard. Mater. 279, 236-243. Fazli, M.M., Soleimani, N., Mehrasbi, M., Darabian, S., Mohammadi, J., Ramazani, A., 2015 Highly cadmium tolerant fungi: their tolerance and removal potential. J. Environ. Health. Sci. Eng. 13, 19.
EP
Fomina, M., Gadd G.M., 2014. Biosorption: current perspectives on concept, definition and application. Bioresour. Technol. 160, 3–14.
Gadd, G.M., 2009. Biosorption: critical review of scientific rationale, environmental importance and
AC C
significance for pollution treatment. J. Chem. Technol. Biotechnol. 84, 13–28.
Gautam, S.P., Bundela, P.S., Pandey, A.K., Jamaluddin, Awasthi, M.K., Sarsaiya, S., 2012. Diversity of Cellulolytic Microbes and the Biodegradation of Municipal Solid Waste by a Potential Strain. Inter J Microbiol Volume Article ID 325907, 12 pages. doi:10.1155/2012/325907
Gola, D., Dey P., Bhattacharya A., Mishra A., Malik A., Namburath M., Ahammad S.Z., 2016. Multiple heavy metal removal using an entomopathogenic fungi Beauveria bassiana. Bioresour. Technol. 218, 388–396. Ghosh, P., Gupta. A., Thakur, I.S., 2015. Combined chemical and toxicological evaluation of leachate from municipal solid waste landfill sites of Delhi, India. Environ. Sci. Pollut. Res. 22 (12), 9148-9158.
ACCEPTED MANUSCRIPT
Hoornweg, D., Tata, P.B., 2012. What a waste. A Global Review of Solid Waste Management. Urban Development Series Knowledge Paper. Public Disclosure Authorized. The World Bank. 5, pp-81. http://documents.worldbank.org/curated/en/302341468126264791/pdf/68135-REVISED-What-aWaste-2012-Final-updated.pdf
RI PT
Kapoor, A., Viraraghavan, T., 1998. Biosorption of heavy metals on Aspergillus niger. Effect of pretreatment. Bioresour. Technol. 63, 109-133.
Koelmel, J., Prasad M.N.V., Velvizhi G., Butti S.K., Mohan S.V., 2016. Metalliferous waste in India and Knoweldge explosion in metal recovery techniques and process for preservation of pollution. Environmental Materials and Waste. http://dx.doi.org/10.1016/B978-0-12-803837-6.00015-9
SC
Lo, Y.C., Cheng C.L., Han Y.L., Chen B.Y., Chang J.S., 2014. Recovery of high-value metals from geothermal sites by biosorption and bioaccumulation. Bioresour. Technol. 160, 182–190
M AN U
Mishra, A., Malik, A., 2014. Novel fungal consortium for bioremediation of metals and dyes from mixed waste stream. Bioresour. Technol. 171, 217-226.
Mosbah, R., Sahmoune, M.N., 2013. Biosorption of heavy metals by Streptomyces species – an overview. Cent. Eur. J. Chem. 11 (9), 1412–1422.
Nongmaithem, N., Roy A., Bhattacharya, P.M., 2016. Screening of Trichoderma isolates for their potential of biosorption of nickel and cadmium. Brazil. J. Microbiol. 47, 305–313. Pandey, A.K., Jamaluddin., Awasthi A.K., Pandey, A., 2013. Biosorption potential of indigenous fungal
TE D
strains for municipal solid waste leachate management in Jabalpur city. J. E. C. E. T. 2 (2), 385-393. Razarinah, W.A.R.W., Zalina, M.N., Abdullah, N., 2014. Treatment of landfill leachate by immobilized Ganoderma australe and crude enzyme. Sci. Asia. 40, 335-339. Salunkhe, R.B., Borase, H.P., Patil, C.D., Patil, S.N., Patil, S.V., 2015. Effect of Different Carbon Sources on
1409-1423.
EP
Morphology and Silver Accumulation in Cochliobolus lunatus. Appl. Biochem. Biotechnol. 177,
Shakya, M., Sharma, P., Meryem, S., Mahmood, Q., Kumar, A., 2015. Heavy Metal Removal from Industrial
AC C
Wastewater Using Fungi: Uptake Mechanism and Biochemical Aspects. J. Environ. Engg. C601500118. DOI: 10.1061/(ASCE)EE.1943-7870.0000983
Silva, L. J., Pinto F.R., Amaral L.A., Garcia-Cruz C.H., 2014. Biosorption of cadmium (II) and lead (II) from aqueous solution using exopolysaccharide and biomass produced by Colletotrichum sp., Desalination
Water Treat. 52 (40-42), 7878-7886.
Sun, Y.M., Horng C.Y., Chang F.L., Cheng L.C., Tian W.X., 2010. Biosorption of lead, Mercury and cadmium ions by Aspergillus terreus immobilized in a natural matrix. Pol J. Microbiol. 59 (1), 37-44.
ACCEPTED MANUSCRIPT
Vijayaraghavan, K., Balasubramanian, R., 2015. Is biosorption suitable for decontamination of metal-bearing wastewaters? A critical review on the state of the art of biosorption processes and future directions. J. Environ. Manage. 160, 283-296. Xu, X., Xia L., Zhu W., Zhang Z., Huang Q., Chen W., 2015. Role of Penicillium chrysogenum XJ-1 in the and
Bioremediation
of
Cadmium.
Front.
Microbiol.
6,
1422.
RI PT
Detoxification
doi:10.3389/fmicb.2015.01422
Yang, Z., Zhou X., Xu L., 2015. Eco-efficiency optimization for municipal solid waste management. J. Clean Prod. 104, 242-249.
Zafar, S., Aqil, F., Ahmad, I., 2007. Metal tolerance and biosorption potential of filamentous fungi isolated
SC
from metal contaminated agricultural soil. Bioresour. Technol. 98, 2557-2561.
Zhang, S., Zhang X., Chang C., Yuan Z., Wang T., Zhao Y, Yang X., Zhang Y., Guixiao La, Wu K., Zhang
M AN U
Z., Li X., 2016. Improvement of tolerance to lead by filamentous fungus Pleurotus ostreatus HAU-2 and its oxidative responses. Chemosphere 150, 33-39.
Zhao, M., Zhang C., Zeng G., Cheng M., Liu Y., 2016. A combined biological removal of Cd2+ from aqueous solutions using Phanerochaete chrysosporium and rice straw. Ecotoxicol. Environ. Safet. 130, 87–92. Zhu, W., Xu. X., Xia. L., Huang. Q., Chen. W., 2016. Comparative Analysis of Mechanisms of Cd2+ and Ni2+
Captions of Figures
TE D
Biosorption by Living and Nonliving Mucoromycote sp. XLC, Geomicrobiol. J. 33 (3-4) 274-282.
Figure 1. Proposed systematic mechanism for biosorption of cadmium heavy metal employing optimized developed fungal consortium. Figure 2. Effect of pH on biosorption of cadmium metal employing developed fungal consortium.
EP
Figure 3. Effect of temperature (0C) on biosorption of cadmium metal employing developed fungal consortium.
AC C
Figure 4. Effect of inoculum concentration (cfu ml−1) of developed fungal consortium on biosorption of cadmium metal Figure 5. Effect of incubation period on biosorption of cadmium metal employing potential fungal strains. Figure 6. Comparative evaluation of biosorption potential by developed fungal consortium and natural flora.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
A systematic illustration of cadmium metal biosorption by indigenous fungi is presented.
•
The optimal ratio of developed fungal consortium removed 72.41% of the cadmium.
TE D
M AN U
SC
Major key mechanisms are evaluated for their role in the biosorption process.
EP
•
The efficiency of biosorption of Cd2+ is depending on the different factor.
AC C
•
RI PT
•
S0959-6526(16)31529-3
DOI:
10.1016/j.jclepro.2016.09.188
Reference:
JCLP 8148
To appear in:
Journal of Cleaner Production
Received Date: 16 June 2016 Revised Date:
11 September 2016
Accepted Date: 23 September 2016
Please cite this article as: Awasthi AK, Pandey AK, Khan J, An eco-friendly approach for minimizing pollution of metal from municipal solid waste leachate in India, Journal of Cleaner Production (2016), doi: 10.1016/j.jclepro.2016.09.188. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
An eco-friendly approach for minimizing pollution of metal from municipal solid waste leachate in India Abhishek Kumar Awasthi a, c * 1, Akhilesh Kumar Pandey a, b, Jamaluddin Khan a a
RI PT
Mycological Research Laboratory, Department of Biological Sciences, Rani Durgavati University, Jabalpur (M.P.), India. b Madhya Pradesh Private Universities Regulatory Commission, Bhopal, India c Department of Microbiology and Biotechnology, V.A.B. College, Chhatarpur, M.P. India 1 Present address: School of Environment, Tsinghua University, Beijing P.R. China 100084
SC
*Corresponding author: A. K. Awasthi, Department of Microbiology, V.A.B. College, Chhatarpur, M.P. India. (M.P.), India. E-mail- [email protected]
Abstract
M AN U
Municipal solid waste (MSW) leachate management has become an important issue worldwide. The present study evaluated the potential of indigenous fungi for biosorption of Cd2+ from leachate. Three isolates—Trichoderma harzianum, Aspergillus niger and Aspergillus flavus were selected based on their ability to exclude the Cd2+ at the preliminary stage. Among these, Trichoderma harzianum was found to be an excellent fungus for cadmium absorption. The highest biosorption rate for Cd2+ was achieved under the conditions: temperature 450C (64.87%), pH 6.0 (64.78%) and
TE D
spore concentration [105cfu ml−1] (64.41%). The obtained data indicated that the developed fungal consortium is very efficient and effective for cadmium removal (72.41%), thereby demonstrating that it is a promising solution for removing toxic metals from MSW leachate.
AC C
1. Introduction
EP
Keywords: Biosorption, Cadmium metal, Indigenous fungi, Municipal solid waste; Waste management.
Municipal solid waste has become the fastest-growing waste stream and its proper collection, recycling and final disposal is one of the most challenging and problematic tasks throughout the world (Yang et al., 2015). The World Bank report estimated that the generation rate of MSW was 0.34 kg/capita/day and 109,589 tonnes/day in 2005. In addition, these figures were expected to rise to 0.7 kg/capita/day and 376,639 tonnes/day by 2025 (Hoornweg and Tata, 2012). The MSW is commonly managed with methods, such as composting, landfill disposal, incineration and open dumping. Among the current management practices, the cheapest—open disposal—is the most common, particularly in developing countries, such as India. However, such
ACCEPTED MANUSCRIPT
practices are a potential threat to the surrounding environment. Rainfall percolating through these open dumping sites triggers biological processes, chemical reactions and physical changes as the MSW degrades, generating a highly contaminated liquid known as leachate. Leachate contains high concentrations of dissolved organic materials, inorganic substances and two categories of heavy
RI PT
metals: essential metals (copper, manganese, zinc and iron), and non-essential metals (cadmium, lead, mercury and nickel). These metals are among the major environmental pollutants because of their high toxicity. However, the exact composition of leachate can vary and depending on the type of waste, climatic conditions, rainfall patterns, hydrological factors and age of the disposal waste
SC
(Ghosh et al., 2015). Numerous physico-chemical methods are commonly employed to remove heavy metals from effluent. These physical and chemical treatment processes are not effective for
M AN U
the management of MSW leachate. In fact, the less ecofriendly technologies are often inefficient, highly expensive and also possess secondary pollution (Fomina and Gadd 2016). Hence, the development of new sustainable and ecofriendly technologies is essential for the treatment of leachate.
Over the past few years, microbial treatment approaches have been attracting more attention, owing to their efficiency, comparative cost-effectiveness and eco-friendliness (Nongmaithem et al.,
TE D
2016; Zhao et al., 2016; Vijayaraghavan and Balasubhaniam 2015). These methods have many advantages, such as easy handling and maintenance (requiring less technical support), higher metal uptake capacity with high treatment rates, minimal sludge production, and high reusability capabilities (Bazrafshan et al., 2016; Fomina and Gadd 2014; Lo et al., 2014). The mechanism of
EP
removing cadmium through microorganisms is a highly complex process because there is competition among various substances, for surface binding sites. The metal biosorption mechanism
AC C
depends on chelation, ion exchange, complexion, adsorption, absorption or micro-precipitation or a combination of these methods (Pandey et al., 2013). The transfer of a metal from outside a cellular membrane into the intracellular space is a metabolism-dependent process governed by a living microbial system. This process is also a microbe’s active defense system, which is potentially capable of tolerating metals. Several microorganisms, including the fungi Pencillium, Rhizopus, Aspergillus and Mucor, as well as yeast and various bacteria, have been used in metal-polluted sites, owing to their continuous enrichment capabilities and highly adaptive nature (Vijayaraghavan and Balasubramanian, 2015; Pandey et al., 2013).
ACCEPTED MANUSCRIPT
Fungi have a bulky biomass and many strains are capable of accumulating either a single heavy metal or multiple metals simultaneously (Mishra and Malik, 2014). The heavy metals removal depends on several factors, such as pH, temperature, incubation period, and inoculum concentration (Koelmel et al., 2016; Chojnacka K., 2010; Sun et al., 2010). The protonation and deprotonation of
RI PT
functional and carboxyl groups play a significant role in the process of metal biosorption (Lo et al., 2014). Mosbah and Sahmoune (2013) stated that, the operational pH range could affect the accessibility of sorption sites on the cell wall surface and the capability of sorption sites to bind metal ions. Thus, these microbes could hypothetically be applied in situ in leachate treatment. Actual
SC
utilization of these strains would require that they have extraordinary tolerance to Cd2+: able to efficiently eliminate it. Yet, in spite of numerous studies on the ability of potential fungi isolates to
M AN U
extract Cd2+, the reaction of fungal strains to this metal has not been thoroughly studied. The objective of this investigation was to assess the practicability of application indigenous fungus from MSW site and development fungal consortia (Trichoderma harzianum, Aspergillus niger and Aspergillus flavus) for the removal of cadmium metal from MSW leachate. The removal efficiency of cadmium metal under diverse conditions (pH, temperature, inoculum concentration and
TE D
incubation period) was examined.
2. Materials and methods
2.1. Sample collection, processing and analysis
The MSW leachate samples were collected from different dumping site around Jabalpur city,
EP
Madhya Pradesh (M.P.) India. During the survey-it was observed that the MSW at site include organic and inorganic matter as well as some of electronic components. The collected leachate
AC C
sample was filtered by Whatman filter paper (Grade 42), before using in the subsequent biosorption experiments or further investigation. The pre-treated and post-treated leachate were also analyzed by Inductive Coupled Plasma Mass Spectrophotometer (ICP-MS) as per Pandey et al. (2013).
2.2. Isolation and identification of indigenous fungal strains
The indigenous fungal isolates were cultivated on potato dextrose agar (PDA) medium through serial dilution method as described by Gautam et al. (2012). The prepared different dilution of leachate sample was inoculated into molten agar medium by using pour plate method under aseptic condition, then the plates were incubated initially for seven days (d) at 28±10C. After desired
ACCEPTED MANUSCRIPT
incubation period, the fungal species were further isolated by single spore culture method (Fig. 1). These isolates were identified on the basis of morphological characteristic, color and appearance of colony and texture, microscopic characteristics (septation of mycelium, shape, diameter and texture of conidia) by slide culture technique and also referred the available manual of principles and
RI PT
practice, (Barnett and Hunter, 1998). These fungal isolates were deposited in the Fungal Germplasm Culture Collection Center (FGCC), M.P. Council of Science and Technology, Jabalpur and also obtained accession number (SI).
SC
Fig. 1. Proposed systematic mechanism for biosorption of cadmium heavy metal employing
M AN U
optimized developed fungal consortium.
2.3. Optimization of environmental condition for fungal growth
The effects of different environmental factors (eg., pH, temperature, inoculum concentration) on the biosorption efficiency of fungal strains in the medium were studied, and control experiments also observed simultaneously. The influence of initial pH on biosorption ability was conducted at a pH range of 3.0 to 8.0, which was adjusted using 0.1 M NaOH and 0.1 M HCl.
TE D
All experiments were carried out in a 250 mL sterilized Erlenmeyer flask containing 150 mL leachate in triplicate. The influence of initial pH on biosorption ability was conducted at a pH range of 3.0 to 8.0, which was adjusted using 0.1 M NaOH and 0.1 M HCl. Batch experiments were performed at different temperature range 28 0C±1 to 60 0C±1 (28 0C, 30 0C, 35 0C, 40 0C, 45 0C, 50 C, 55 0C, 60 0C) with respect to diverse incubation period e.g., 7d, 14d, 21d, 28d, 35d. The
EP
0
cadmium metal concentration after biosorption in leachate were analyzed by using Inductive Coupled Plasma Mass Spectrophotometer (ICP-MS). In the same time, leachate sample does not
AC C
contain fungal spores served as control also studies. The data obtained from the independent experiments were analyzed by used SPSS 15 software. The biosorption potential i.e., the amount of cadmium metal ion (mg/g-1) was calculated by using following equation (1):
Where, ܳ- mg of metal ion biosorbed per g ݅ܥ- Initial metal ions concentration (mg/l) ݂ܥ- Final metal ion concentration (mg/1)
Q =
[Ci − Cf ]v ………………………………………….(1) s
ACCEPTED MANUSCRIPT
ݏ- Wet biomass ݒ- Volume of reaction mixture
3. Results and discussion
RI PT
3.1 Isolation and identification of heavy-metal-tolerant fungi Thirty indigenous fungal isolates were isolated from leachate samples of a municipal solid waste dumping site using standard techniques (Gautam et al., 2012). The effects of cadmium concentration on the growth and morphology of the fungi were also evaluated in a broth culture. Various isolated
supplementary information (SI).
M AN U
3.2 Screening of fungal isolates for tolerance to Cd2+ metal
SC
fungi were identified based on morphological characteristics and these are presented in the
The observed fungal growth of the all the fungal strains, relative to each other, on solid PDA media supplemented with different Cd2+ concentrations. The indigenous fungal isolates showed an ability to grow very rapidly and to cover the entire surface of a petriplate containing PDA Cd2+ concentration levels of less than 0.5 mg/L within a 10-day incubation period, while the remaining strain exhibited very slow growth at a concentration level of 1 mg/L (data not show). At the
TE D
concentration level of more than 1.5 mg/L, three strains—Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus (FGCC#A03)—showed very limited growth. However, no development occurred at all for the other strains, and even for these three strains, the growth rate declined with the addition of extra Cd2+. Nevertheless, the growth of these indigenous
EP
fungi indicated that these strains had potential biosorption ability for Cd2+, and could be used for further experiments.
AC C
These indigenous fungal strains—Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus (FGCC#A03)—showed higher Cd2+-metal tolerance than other identified fungi. Similarly, Pandey et al. (2013) found that indigenous fungal isolates had impressive abilities to remove metal from MSW leachate. The indigenous fungal isolates have more tolerance potential (Pandey et al., 2013). Actually, the removal of heavy metals by microbes seems to depend on both the metal and the fungal species. Among the indigenous strains, we found the two isolates Aspergillus and Trichoderma sp. to be highly Cd2+-tolerant and able to survive in cultures with more than 1.5 mg/L. Our outcomes revealed, that the biosorption ability decreased with higher Cd2+ concentration. In the same time- also noticed that a rise above the initial metal level resulted
ACCEPTED MANUSCRIPT
decrease in the metal exclusion ability of the fungi. It meant that the heavy metals may be harmful to cells when substantial adsorption is established on cell walls. In living fungi, the leading source of metal accumulation is through intracellular uptake via the cell membrane. An efflux tool could also function at a specific metal concentration to prevent further
RI PT
accumulation. The remarkable metal tolerance of fungi is directly related to fungal survival and growth in metal-polluted leachate, and our potential fungal isolates showed considerable metal resistance potential. While, this study did not examine the mechanism of tolerance, these outcomes explained the possible applicability of employing indigenous fungal isolates for cadmium exclusion:
SC
a particularly significant finding for any sites with very high levels of Cd2+ pollution. Similar findings have been reported by many authors. Fazli et al. (2015) suggested that A. versicolor could
M AN U
accumulate Cd2+ at the concentration of 7 mg/g mycelium, Paecilomyces sp. at 5.878 mg/g, Microsporum at 5.07 mg/g, and Trichoderma sp. at 4.55 mg/g. A number of other authors have also studied fungal tolerance to heavy metals (Carrillo and Gonzalez et al., 2012).
3.3 Optimum biosorption conditions of fungal strains
Although numerous microbes have the ability to bind with heavy metals, only a few of them have
TE D
shown excellent metal exclusion through the binding process. Yet maximum metal removal rates can be achieved when optimize the environmental conditions. Such optimal conditions can be established through biosorption experiments. In this study, the optimal conditions for Cd2+ metal exclusion (pH, temperature and incubation period) were determined for highly tolerant isolates of
AC C
(FGCC#A03).
EP
Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus
3.3.1 Effect of pH on metal biosorption Heavy metal biosorption potential can be affected by pH because the acidic level of the metal solution affects the surface binding charge of the fungal biosorbent, and also influences the solubility of the metal. As shown in the Fig. 2, a pH value above 6.0 caused the precipitation of Cd2+. These findings suggested that the Cd2+ biosorption by Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01), and Aspergillus flavus (FGCC#A03) increased steadily as pH rose, reaching its maximum at pH 6.0 and then declining as the pH fell to 8.0. This reduction at pH>6 might be caused by complexation with soluble organic ligands (Gola et al., 2016). Low pH (pH<4) also decreased the
ACCEPTED MANUSCRIPT
biosorption rate, with lower pH values corresponding to lower biosorption rates (Fan et al., 2014). This pattern occurred in all three of the fungi strains—Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01) and Aspergillus flavus (FGCC#A03). The biosorption of metal is highly correlated with the pH of the solution is that it can impact the
RI PT
metals’ chemistry and the activity of functional groups (carboxylate, phosphate, and amino groups) on the cell wall, because of competition between metal ions and binding sites (Bazrafshan et al., 2016; Nongmaithem et al., 2016). Studies conducted by Fan et al. (2014) suggested that the biosorption ability of metal is lower at lower pH levels because of hydrogen ions’ competition with
SC
metal ions present on the fungal cell wall sites. However, metal precipitates at higher pH levels, inhibiting the contact of metal with most fungal biomass (Razarinah et al., 2014)—a phenomenon
M AN U
that accounts for the drop in biosorption rate as pH increases above 6.0.
Fig. 2. Effect of pH on biosorption of cadmium metal employing developed fungal consortium.
3.3.2. Effect of temperature on metal biosorption
Temperature is another important factor in the biosorption process. As shown in the Fig. 3, the
TE D
biosorption of Cd2+ removal increased from 33.2% to 63.98% as the temperature rose from 28 0C to 45 0C, and then abruptly decreased between 45 0C and 60 0C, for all the inoculum concentrations of the three individual fungal strains [Trichoderma harzianum (FGCC#A29), Aspergillus flavus (FGCC#A01), and Aspergillus niger (FGCC#A03)], although the maximum Cd2+ biosorption was
EP
51.01% at 50 0C for the Trichoderma harzianum (FGCC#A29). Hence our investigation confirmed that temperature plays a significant role in the biosorption process (Lo et al., 2014; Ahalya et al.,
AC C
2003). Pandey et al. (2013) suggested that maximum metal uptake activity is correlated with higher temperature because high temperature increases the affinity of metal-binding sites on fungal cell walls. Nevertheless, room temperature is suitable—although not optimal—for an effective biosorption process. This correlation between biosorption ability and higher temperature needs further, more detailed investigation. Fig. 3. Effect of temperature (0C) on biosorption of cadmium metal employing developed fungal consortium.
ACCEPTED MANUSCRIPT
3.3.3. Effect of inoculum concentration on metal biosorption The most frequently isolated indigenous fungal isolates from the leachate sample were, in order: Aspergillus sp., Alternaria sp., Curvularia sp., Fusarium sp., Mucor sp., Rhizopus sp. Of these indigenous isolates only three fungal strains were selected based on their potential capacity for metal
RI PT
biosorption at various inoculum concentrations. The fungal isolates Cladosporium oxysporum (FGCC±A10), Chaetomium globosum (FGCC#A11) and Cunningmella echinulate (FGCC#A12) showed relatively low levels of cadmium biosorption compared to the other isolates. The fungal isolates showing the highest cadmium metal tolerances through ionic association among
SC
extracellular polysaccharides, proteins and chitins, were in order: Trichoderma harzianum (FGCC#A29), Aspergillus niger (FGCC#A01) and Aspergillus flavus (FGCC#A03). Other fungi,
M AN U
such as Ganoderma austral, Aspergillus terreus, A. lentulus and Rhizopus oryzae have been reported on by researchers studying MSW leachate biosorption (Mishra and Malik, 2014; Razarinah et al., 2014). Furthermore, during the standardization process, it was noted that maximum biosorption of Cd2+ metal was accomplished using Trichoderma harzianum FGCC#A29 (51.01%) followed by Aspergillus niger FGCC#A01, (40.62%) and A. flavus FGCC#A03 (37.65%) and that biosorption activity can be affected by, inoculum concentration (Fig. 4), temperature pH and incubation time
TE D
(Fig 5).
cadmium metal
EP
Fig. 4. Effect of inoculum concentration (cfu ml−1) of developed fungal consortium on biosorption of
Fig. 5. Effect of incubation period on biosorption of cadmium metal employing potential fungal
AC C
strains.
3.3.2. Development of fungal consortium It was observed that excessive cadmium concentration was able to inhibit fungal growth during the lag phases. A single fungal strain, cannot effectively treat leachate. It must be tested in a synergistic relationship with other potential fungal isolates, and then a fungal consortium can be developed based on their synergistism performance. In this study, it was found that the developed consortium performed well for the biosorption of Cd2+ (72.41% and 30.34% by fungal consortia and natural flora respectively) from MSW leachate at pH 6.0±1; 450C; 35d), as shown in the fig. 6.
ACCEPTED MANUSCRIPT
Hence, it can be assumed that a standard combination of these three fungal isolates would be helpful in developing an effective leachate treatment (Fig. 5). Therefore, this studied indicates synergistic relationship in order to better understand the developed fungal consortium. Bazrafshan et al. (2016) stated that metal biosorption directly related to valency and atomic number, and that Cd2+ had a
RI PT
greater affinity to the binding sites of our developed fungal consortium than to any of the individual fungal strains. The biosorption potential percentage of Cd2+ using the developed consortium was higher than when using the individual fungal isolates, under all growth conditions. Nevertheless, an optimal fungal growth environment was an essential factor for increasing the percentage of metal
SC
biosorption.
The efficacy of indigenous fungi in the management of heavy-metal-contaminated MSW
M AN U
leachate is a promising approach because the mechanism involves both extracellular adsorption and intracellular accumulation or absorption: in other words, biosorption. This promising strategy— using fungi for the biosorption of Cd2+ metal—has been previously reported (Bazrafshan et al., 2016; Zhu et al., 2016; Silva et al., 2014;). In order to achieve these results, a fungus must have a very high tolerance level to heavy metals. Many fungal strains were tested and failed to propagate because of the toxicity of the heavy metals, making them unsuitable. Zhang et al. (2016) stated that
TE D
acclimatization is one possible way to enhance a microorganism’s resistance to heavy metals; they used a wild strain of Pleurotus ostreatus HAU-2, inoculated with media containing different concentrations of metal (Pb), and found that after 90 days of incubation, the fungus’s ability to survive the metal’s toxicity was enhanced significantly. This result indicates that using indigenous
isolates.
EP
fungi for metal removal from leachate could be more efficient than using non-indigenous fungal
AC C
Many studies have shown that heavy metal biosorption employing fungi have great ability because their cell walls are composed of proteins, lipids, and polysaccharides—different functional groups that are able to bind to metal (Vijayaraghavan and Balasubramanian, 2015). A cell wall made of chitin and chitosan (mixed polysaccharides) plays an important role in metal binding. Hence fungi, such as Aspergillus niger can remove metals with its spores and can therefore act as an excellent biosorbent for metals. Cd2+ metal biosorption employing several different fungi, at various concentration levels, has been well documented by several researchers (Kapoor and Viraraghavan, 1998). Exact results have varied, however, among different investigations, owing to the type of microbes used, the inoculum concentration, temperature, and pH as well as the experimental
ACCEPTED MANUSCRIPT
methods. For instance, Xu et al. (2015) studied cadmium detoxification using Penicillium chrysogenum XJ-1 with a method that included biosorption, cellular sequestration, and antioxidant defense, and their findings suggested that Cd2+ metal had been effectively eliminated from contaminated soils (at the concentration 5–50mg kg−1). Still, this fungal strain needs more study,
RI PT
possibly by mixing it with compost or biochar for improving the plant growth and development. Exposure to cadmium can cause modifications to fungal morphology, in all the indigenous fungal strains. Some researchers have suggested that it forming colorful mycelia due to admits of heavy metals on media (Shakya et al., 2015). Vijayaraghavan and Balasubhaniam (2015)
SC
emphasized that most of the studies carried out previously had certain constraints for successful metal removal, such as only working with synthetic solutions.
M AN U
Fig. 6. Comparative evaluation of biosorption potential by developed fungal consortium and natural flora.
Many researchers have carried out detailed study on Cd2+ biosorption, using various fungal strains, such as Aspergillus & Rhizopus [2.72-2.91 mg/g-1 & 2.72 mg/g-1] (Zafar et al., 2007) and Aspergillus niger [1.31 mg/g-1] (Kapoor and Viraraghavan, 1998). One of their findings was that excessive concentration levels of cadmium metal could cause discoloration of the fungus, such as
TE D
changing pink to white, as in the case of Paecilomyces sp. and Paecilomyces sp. genera. Researchers have also reported pigment production in the fungal cell and in cell-free media, together with metal ion precipitation on cell walls. This production may act as a preliminary defense, to protect the
EP
protoplast from metal toxicity (Fazli et al., 2015). Microbes sometimes produce metal-chelating proteins, such as metallothionein and glutathione, which are essential for the reduction of cadmium toxicity. After saturating the binding sites of the
AC C
fungal cell walls, the metal crosses into the vacuoles, and is then chelated with different types of organic acid (Shakya et al., 2015). The protonation and deprotonation of functional groups also make significant contributions to the biosorption process, because pH may alter the approachability of sorption sites for binding metal ions to a specific surface site. Although higher metal removal could be accomplished at either a neutral or an alkaline range of pH, that reflects the deprotonation of carboxyl group (Pandey et al., 2013). Some other researchers have also determined that different metal concentrations can affect fungal growth rates (Aspergillus carbonarius, Fusarium sp. and Penicillium). It may be that the metals affect their enzymatic activity, resulting in a decrease in the
ACCEPTED MANUSCRIPT
production of conidiophores (Xu et al., 2015). Carrillo and Gonzalez (2012) found that the indigenous fungus Scleroderma citrinum proved to have a good tolerance level for Cd2+ (78%-95%) and that this tolerance depended not only on the metal concentration but also on whether the strain is indigenous.
RI PT
Some authors have proposed that the main mechanism of biosorption of metal is related to ion exchange (Ahalya et al., 2003). Microbes vary in their compositions of membrane lipid cell walls, and different growth phases of cell membranes may directly affect their susceptibilities to toxic substances (Fan et al., 2014). In other words, the fungal cell wall significantly contributes to the
SC
adsorption of Cd metal ions, and the–OH and–C=O groups on their cell surface are responsible for the Cd2+ binding. The cell wall is the first line of defense and also protects the protoplast from metal
M AN U
poisoning for short periods of metal threat. When the saturation stage is reached, most intracellular metals have a tendency to be transported to vacuoles and then chelated with different organic acids: e.g., citric, oxalic, etc. to accomplish metal compartmentalization. Toxic metal deposition on cell walls and vacuolar compartmentalization of microbes are well known to be metal detoxification tools. Thus, intracellular compartmentalization could play a key role in the decontamination and tolerance of Cd2+ in the fungal strains.
TE D
Salunkhe et al. (2015) suggested that morphology, such as fibrousness, hyphae, pelletization, and clumping of filamentous fungi play crucial roles in the uptake of metabolites and enzyme production. Zhu et al. (2016) stated that living cells always have better capabilities as a biosorbent for metal (Ni2+, Cd2+, Cu
2+
and Cr
3+
), than non-living substances, because the available binding
EP
sites are very limited on non-living absorbent, and excess metal ions could neither bind on it nor be removed from the solution. The biosorption capacity of living cells, however, might be improved
AC C
with the application of more energy, so that they could deal with increases in the metal concentration.
The main benefits of biosorption are that it is cost effective, non-invasive, sustainable, and more ecofriendly than conventional technologies, such as physical, chemical, incineration etc. Furthermore, this process can be made site-specific, reducing health risk, and it permanently detoxifies the waste, eliminating long-term liability. It could also be combined with physical or chemical technologies. The process can be varied by using different types of biosorbents, such as microbial biomass (i.e., bacteria, fungi and yeast), algal biomass, proteins, and other biomaterials.
ACCEPTED MANUSCRIPT
Furthermore, the strains could be used as successful microbial agents for solving the problem of cadmium metal pollution in the natural environment. As shown in the above-discussed studies, the developed fungal consortium is promising candidate for the detoxification of heavy metal from MSW leachate. Yet despite its many
RI PT
advantages, many challenges still remain for biosorption. For example, Vijayaraghvan and Balasubramanium (2015) have suggested that significant work is still needed to improve biosorption efficiency. The dearth of operational environments and the need for optimization could restrict the practical and effective application of developed consortia in the transformation process. Chojnacka
SC
(2010) believed that, this method is needed to supplement the external source of energy (eg. Sucrose) to growing cells. Though, if the suitable fungal strains are identified, it is promising to
M AN U
recommend a self-replenishing method whereby microbial cells which absorb or adsorb the contaminants (either organic compounds or inorganic ions). Although, this approach has not been explored systematically, yet. Because there might be a demand for processing vast amounts of heavy metals having a toxicological impact on the surrounding environment. Gadd (2009) said that, the limitations such as, shorter life time of biosorbents compared to conventional approach. Large-scale implementation of biosorption is not currently practical, and further study is necessary, to deal with
TE D
heavy-metal contamination of the environment.
The next step is to determine exactly how the growth and development of these fungal isolates can be extended to remediate the cadmium metal-polluted soils of the Jabalpur MSW site. More
4. Conclusion
EP
advanced research is warranted, to find more eco-friendly disposal methods.
This research provided an opportunity to evaluate the biosorption potential of indigenous fungal
AC C
isolates. Results clearly revealed that inoculation of potential strains in leachate can effectively and efficiently accelerate the biosorption mechanism and remove cadmium metal, while natural flora showed weak performance. The optimal conditions were: spore concentration 10-5, pH 6.0±1 and temperature 450C. These conditions achieved significant biosorption (72.41%) for cadmium metal. This method is an ecofriendly, cost-effective and energy-saving approach to leachate treatment. More research is needed, however, before implementing the technology on a large scale.
Acknowledgements
ACCEPTED MANUSCRIPT
The authors are thankful Head, Department of Biological Science, R.D. University, Jabalpur for providing laboratory facilities and also thankful to Municipal Corporation Jabalpur (M.P.) India for their support.
RI PT
Appendix A. Supplementary information (SI) attached in this manuscript. References
Ahalya, N., Ramachandra T.V., Kanamadi R.D., 2003. Biosorption of Heavy Metals. Res. J. Chem. Environ. 7(4), 71-78.
Barnett, H.L., Hunter B.B., 1998. Illustrated genera of imperfect fungi (3rd Edn.) ABS Press. The Am. Phyto.
SC
Soc., Minnesota.
Bazrafshan, E., Zarei A.A., Mostafapour F.K., 2016. Biosorption of cadmium from aqueous solutions by Trichoderma fungus: kinetic, thermodynamic and equilibrium study. Desalination Water Treat. 57
M AN U
(31), 14598-14608.
Carrillo, G.R., Gonzalez-Chavez Mdel, C., 2012. Tolerance to and accumulation of cadmium by the mycelium of the fungi Scleroderma citrinum and Pisolithus tinctorius. Biol. Trace. Elem. Res. 146, 388-395.
Chojnacka, K., 2010. Biosorption and bioaccumulation e the prospects for practical applications. Environ. Inter. 36, 299-307.
TE D
Fan, J., Okyay, T.O., Rodrigues, D.F., 2014. The synergism of temperature, pH and growth phases on heavy metal biosorption by two environmental isolates. J. Hazard. Mater. 279, 236-243. Fazli, M.M., Soleimani, N., Mehrasbi, M., Darabian, S., Mohammadi, J., Ramazani, A., 2015 Highly cadmium tolerant fungi: their tolerance and removal potential. J. Environ. Health. Sci. Eng. 13, 19.
EP
Fomina, M., Gadd G.M., 2014. Biosorption: current perspectives on concept, definition and application. Bioresour. Technol. 160, 3–14.
Gadd, G.M., 2009. Biosorption: critical review of scientific rationale, environmental importance and
AC C
significance for pollution treatment. J. Chem. Technol. Biotechnol. 84, 13–28.
Gautam, S.P., Bundela, P.S., Pandey, A.K., Jamaluddin, Awasthi, M.K., Sarsaiya, S., 2012. Diversity of Cellulolytic Microbes and the Biodegradation of Municipal Solid Waste by a Potential Strain. Inter J Microbiol Volume Article ID 325907, 12 pages. doi:10.1155/2012/325907
Gola, D., Dey P., Bhattacharya A., Mishra A., Malik A., Namburath M., Ahammad S.Z., 2016. Multiple heavy metal removal using an entomopathogenic fungi Beauveria bassiana. Bioresour. Technol. 218, 388–396. Ghosh, P., Gupta. A., Thakur, I.S., 2015. Combined chemical and toxicological evaluation of leachate from municipal solid waste landfill sites of Delhi, India. Environ. Sci. Pollut. Res. 22 (12), 9148-9158.
ACCEPTED MANUSCRIPT
Hoornweg, D., Tata, P.B., 2012. What a waste. A Global Review of Solid Waste Management. Urban Development Series Knowledge Paper. Public Disclosure Authorized. The World Bank. 5, pp-81. http://documents.worldbank.org/curated/en/302341468126264791/pdf/68135-REVISED-What-aWaste-2012-Final-updated.pdf
RI PT
Kapoor, A., Viraraghavan, T., 1998. Biosorption of heavy metals on Aspergillus niger. Effect of pretreatment. Bioresour. Technol. 63, 109-133.
Koelmel, J., Prasad M.N.V., Velvizhi G., Butti S.K., Mohan S.V., 2016. Metalliferous waste in India and Knoweldge explosion in metal recovery techniques and process for preservation of pollution. Environmental Materials and Waste. http://dx.doi.org/10.1016/B978-0-12-803837-6.00015-9
SC
Lo, Y.C., Cheng C.L., Han Y.L., Chen B.Y., Chang J.S., 2014. Recovery of high-value metals from geothermal sites by biosorption and bioaccumulation. Bioresour. Technol. 160, 182–190
M AN U
Mishra, A., Malik, A., 2014. Novel fungal consortium for bioremediation of metals and dyes from mixed waste stream. Bioresour. Technol. 171, 217-226.
Mosbah, R., Sahmoune, M.N., 2013. Biosorption of heavy metals by Streptomyces species – an overview. Cent. Eur. J. Chem. 11 (9), 1412–1422.
Nongmaithem, N., Roy A., Bhattacharya, P.M., 2016. Screening of Trichoderma isolates for their potential of biosorption of nickel and cadmium. Brazil. J. Microbiol. 47, 305–313. Pandey, A.K., Jamaluddin., Awasthi A.K., Pandey, A., 2013. Biosorption potential of indigenous fungal
TE D
strains for municipal solid waste leachate management in Jabalpur city. J. E. C. E. T. 2 (2), 385-393. Razarinah, W.A.R.W., Zalina, M.N., Abdullah, N., 2014. Treatment of landfill leachate by immobilized Ganoderma australe and crude enzyme. Sci. Asia. 40, 335-339. Salunkhe, R.B., Borase, H.P., Patil, C.D., Patil, S.N., Patil, S.V., 2015. Effect of Different Carbon Sources on
1409-1423.
EP
Morphology and Silver Accumulation in Cochliobolus lunatus. Appl. Biochem. Biotechnol. 177,
Shakya, M., Sharma, P., Meryem, S., Mahmood, Q., Kumar, A., 2015. Heavy Metal Removal from Industrial
AC C
Wastewater Using Fungi: Uptake Mechanism and Biochemical Aspects. J. Environ. Engg. C601500118. DOI: 10.1061/(ASCE)EE.1943-7870.0000983
Silva, L. J., Pinto F.R., Amaral L.A., Garcia-Cruz C.H., 2014. Biosorption of cadmium (II) and lead (II) from aqueous solution using exopolysaccharide and biomass produced by Colletotrichum sp., Desalination
Water Treat. 52 (40-42), 7878-7886.
Sun, Y.M., Horng C.Y., Chang F.L., Cheng L.C., Tian W.X., 2010. Biosorption of lead, Mercury and cadmium ions by Aspergillus terreus immobilized in a natural matrix. Pol J. Microbiol. 59 (1), 37-44.
ACCEPTED MANUSCRIPT
Vijayaraghavan, K., Balasubramanian, R., 2015. Is biosorption suitable for decontamination of metal-bearing wastewaters? A critical review on the state of the art of biosorption processes and future directions. J. Environ. Manage. 160, 283-296. Xu, X., Xia L., Zhu W., Zhang Z., Huang Q., Chen W., 2015. Role of Penicillium chrysogenum XJ-1 in the and
Bioremediation
of
Cadmium.
Front.
Microbiol.
6,
1422.
RI PT
Detoxification
doi:10.3389/fmicb.2015.01422
Yang, Z., Zhou X., Xu L., 2015. Eco-efficiency optimization for municipal solid waste management. J. Clean Prod. 104, 242-249.
Zafar, S., Aqil, F., Ahmad, I., 2007. Metal tolerance and biosorption potential of filamentous fungi isolated
SC
from metal contaminated agricultural soil. Bioresour. Technol. 98, 2557-2561.
Zhang, S., Zhang X., Chang C., Yuan Z., Wang T., Zhao Y, Yang X., Zhang Y., Guixiao La, Wu K., Zhang
M AN U
Z., Li X., 2016. Improvement of tolerance to lead by filamentous fungus Pleurotus ostreatus HAU-2 and its oxidative responses. Chemosphere 150, 33-39.
Zhao, M., Zhang C., Zeng G., Cheng M., Liu Y., 2016. A combined biological removal of Cd2+ from aqueous solutions using Phanerochaete chrysosporium and rice straw. Ecotoxicol. Environ. Safet. 130, 87–92. Zhu, W., Xu. X., Xia. L., Huang. Q., Chen. W., 2016. Comparative Analysis of Mechanisms of Cd2+ and Ni2+
Captions of Figures
TE D
Biosorption by Living and Nonliving Mucoromycote sp. XLC, Geomicrobiol. J. 33 (3-4) 274-282.
Figure 1. Proposed systematic mechanism for biosorption of cadmium heavy metal employing optimized developed fungal consortium. Figure 2. Effect of pH on biosorption of cadmium metal employing developed fungal consortium.
EP
Figure 3. Effect of temperature (0C) on biosorption of cadmium metal employing developed fungal consortium.
AC C
Figure 4. Effect of inoculum concentration (cfu ml−1) of developed fungal consortium on biosorption of cadmium metal Figure 5. Effect of incubation period on biosorption of cadmium metal employing potential fungal strains. Figure 6. Comparative evaluation of biosorption potential by developed fungal consortium and natural flora.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
A systematic illustration of cadmium metal biosorption by indigenous fungi is presented.
•
The optimal ratio of developed fungal consortium removed 72.41% of the cadmium.
TE D
M AN U
SC
Major key mechanisms are evaluated for their role in the biosorption process.
EP
•
The efficiency of biosorption of Cd2+ is depending on the different factor.
AC C
•
RI PT
•