- Email: [email protected]
Bioleaching of manganese by Aspergillus sp. isolated from mining deposits
Accepted Manuscript Bioleaching of manganese by Aspergillus oryzae isolated from mining deposits
Sansuta Mohanty, Shreya Ghosh, Sanghamitra Nayak, Alok Prasad Das PII:
S0045-6535(16)31879-3
DOI:
10.1016/j.chemosphere.2016.12.136
Reference:
CHEM 18592
To appear in:
Chemosphere
Received Date:
18 October 2016
Revised Date:
23 December 2016
Accepted Date:
27 December 2016
Please cite this article as: Sansuta Mohanty, Shreya Ghosh, Sanghamitra Nayak, Alok Prasad Das, Bioleaching of manganese by Aspergillus oryzae isolated from mining deposits, Chemosphere (2016), doi: 10.1016/j.chemosphere.2016.12.136
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ACCEPTED MANUSCRIPT Highlights 1. It elucidates the mechanism of manganese bioleaching by Aspergillus oryzae and the comprehensive factors that emphasize the selection of manganese recovery technique. 2. Includes the understanding of alteration of fungal protein expression in response to Mn bioleaching.
3. Elemental and mineralogical studies of the ore sample were carried out via microscopic analysis, SEM, EDX, XRD and FITR to access the basic arrangement of preferred elements and functional groups.
ACCEPTED MANUSCRIPT
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Bioleaching of manganese by Aspergillus oryzae isolated from mining deposits
2
Sansuta Mohantya, Shreya Ghosha, Sanghamitra Nayak a and Alok Prasad Dasb*
3 4 5
aBioengineering
& Biomineral Processing Laboratory, Centre of Biotechnology,
Siksha O Anusandhan University, Khandagiri Square, Bhubaneswar, India bDepartment
of Chemical and Polymer Engineering, Tripura University (A Central
6
University), Suryamaninagar, Tripura
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Email: [email protected]
8 9 10 11 12 13 14 15 16
Corresponding Author: Dr. Alok Prasad Das*
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Department of Chemical and Polymer Engineering, Tripura University (A Central University),
18
Suryamaninagar, Tripura.
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Email: [email protected]
20
Phone: +919178581814
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Abstract
26
A comprehensive study on fungus assisted bioleaching of manganese (Mn) was carried out to
27
demonstrate Mn solubilization of collected low grade ore from mining deposits of Sanindipur,
28
Odisha, India. A native fungal strain MSF 5 was isolated and identified as Aspergillus oryzae by
29
Inter Transcribed Spacer (ITS) sequencing. The identified strain revealed an elevated tolerance
30
ability to Mn under varying optimizing conditions like initial pH (2, 3, 4, 5, 6, 7), carbon sources
31
(dextrose, sucrose, fructose and glucose) and pulp density (2 %, 3 %, 4 %, 5 % and 6 %).
32
Bioleaching studies carried out under optimized conditions of 2% pulp density of Mn ore at pH
33
6, temperature 37 0C and carbon dosage (dextrose) resulted with 79 % Mn recovery from the ore
34
sample within 20 days. SEM-EDX characterization of the ore sample and leach residue was
35
carried out and the micrographs demonstrated porous and coagulated precipitates scattered
36
across the matrix. The corresponding approach of FTIR analysis regulating the Mn oxide
37
formation shows a distinctive peak of mycelium cells with and without treated Mn, resulting
38
with generalized vibrations like MnOx stretching and CH2 stretch. Thus, our investigation
39
endeavors’ the considerate possible mechanism involved in fungal surface cells onto Mn ore
40
illustrating an alteration in cellular Mn interaction.
41 42
Keywords: Bioleaching, Aspergillus oryzae, Optimization, Protein profiling, SEM, FTIR
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1. Introduction
49
Indiscriminate exploration of heavy metal with major environmental setbacks is one of the
50
major challenges of widespread mining (Bing et al., 2012; Ghosh et al., 2015). The liberation
51
of toxic heavy metals into the atmosphere through industrial effluents is the foremost concern.
52
Mn mining operations deeply provide a profitable advancement although at the same time it
53
depreciates the environment through metal pollutants from mining. Widespread mining and
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random disposal of mining wastes are principally responsible for environmental contamination.
55
The estimated amount of solid waste generated is about 7-8 million tones in the form of
56
disposed minerals, waste sources and lean grade ore to ensuing in ecological pollution (Akcil et
57
al., 2015). As manifested from the current study, Mn contaminated soils cover common metal
58
contaminants in low and high concentration of different types of metals as pollutants.
59
Integration of heavy metal contaminants in environment generally leads to variations in
60
biochemical speciation. However, it is a significant aspect to work on the metal bioavailability
61
onto the toxicity of a heavy metal that are largely depended on the influences of metal ions.
62
(Roane and Kellogg, 1996).
63
The mineral bioleaching ability of Mn solubilizing microorganisms illustrates an essential
64
part for further exploration of this metal from various sources (Ghosh et al., 2015). Recent study
65
reported that (Vera et al., 2013) examined that the oxidation of Mn may involve the alteration of
66
various proteins for regulation of Mn solubilization focusing the induction of insoluble Mn
67
(Myers et al., 2003) oxides forming soluble Mn(III) complex. However, the response of Mn
68
solubilization occurs naturally through enzymatic activity involving enzymes like multicopper
69
oxidase (MCO), cellobiose dehydrogenase (CDH), Manganese peroxidase (MnP), confirming
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the biotransformation of protein patterns of different fungal strains (Hakala et al., 2006;
71
Podorozhko et al., 2008).
72
On considering the effective Mn solubilization by fungal mycelium, our present study aims
73
(1) in optimizing different parameters like initial pH (2, 3, 4, 5, 6, 7), carbon sources (dextrose,
74
sucrose, fructose and glucose) and pulp density (2%, 3%, 4%, 5% and 6%), (2) in determining
75
the Mn leaching efficiency of the isolated fungal strain Aspergillus oryzae MSF-5 under
76
optimized conditions, (3) in ascertaining the changes in the ore composition through FTIR
77
analysis. Thus, our research also endeavors the understanding of alteration of fungal protein
78
expression in response to Mn bioleaching.
79
2. Material and methods
80
2.1 Mineral sample and culture conditions
81
The lean grade Mn ore samples were collected from Sanindipur mines from Barbil of
82
Kendhujhar district, Odisha, India. The samples were finely crushed and grounded, then sieved
83
in various size fractions for leaching experiments. The crushed samples were subjected to acid
84
digestion and examined for estimating the percentage of Mn content present in the ore.
85
Mineralogical analysis of the composed Mn ore sample was examined to access the quality
86
grade. The physico-chemical parameters of the ore sample like size of the particle, TDS, TDS,
87
conductivity, pH was examined. Elemental and mineralogical studies of the ore sample were
88
carried out via microscopic analysis, SEM, EDX (JEOL JSM-6330F), XRD analysis. FITR was
89
used to access the basic arrangement of functional groups.
90 91 92
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2.2. Fungal strain and medium concentrations
94
A fungal species capable of tolerating Mn in high concentration was isolated from the ore
95
assembled from Mn dumping areas of Sanindipur mines. Based on biochemical analysis and
96
ITS
97
GGAAGTAAAAGTCGTAACAAGG-3’) and ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’)
98
the isolated fungal species was classified as Aspergillus oryzae MSF 5 (with accession number
99
KP309810). Initial culturing of the fungal strain was done on potato dextrose agar (PDA) plates
100
at 37 0C until mycelium was observed covering most of the plate surface. Fungal colonies
101
obtained from mining low grade ore were maintained onto an optimized medium i.e. minimal
102
salt medium (MSM) with concentrations like 0.5 % (w/v) Mn: yeast extract - 0.5 g/L, Dextrose
103
- 10,Ammonium sulphate (NH4SO4) - 0.5 g/L, Magnesium sulphate (MgSO4.7H2O) - 0.1 g/L,
104
Dipotassium hydrogen phosphate (K2HPO4) - 1.5 g/L, Sodium chloride (Nacl) - 0.2 g/L, Agar –
105
2 %, pH adjusted to 6.8 as discussed in our previous paper (Mohanty et al., 2016).
(internal
transcribed
spacer)
sequencing
using
primers
ITS-5
(5’-
106 107
2.3. Optimization of Mn bioleaching under different parameters
108
As Mn bioleaching is statistically altered by different parameters, the solubilization of Mn by
109
fungal strains was optimized under several parameters like Mn concentration, pH and sugar
110
(carbon dosage) to regulate the conditions for effective Mn bioleaching via fungal strains. The
111
effect of parameters like pH (2, 3, 4, 5, 6, 7), initial Mn concentration (50, 100, 200, 500 mg/L)
112
and carbon sources (dextrose, sucrose, fructose and glucose) on Mn bioleaching were
113
investigated for a period of 21 days. All the reduction experiments were carried in MSM
114
containing Dextrose-10.0 g/L, yeast extract-0.5 g/L, Ammonium sulphate (NH4SO4)- 0.5 g/L,
115
Magnesium sulphate MgSO4.7H2O - 0.1 g/L, Sodium chloride (Nacl) - 0.2 g/L, Dipotassium
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hydrogen phosphate (K2HPO4) - 1.5 g/L (Pradhan et al., 2005), pH adjusted to 6 with 10 ml of
117
fungal inoculum of A. oryzae and 2 % (w/v) of Mn concentration at pH 6. The error bars were
118
put on the graph obtained by the standard deviation.
119 120
2.3.1. Effects of pH
121
The significant effect of pH on Mn bleaching was studied in the range of pH 2 to 7. The pH was
122
adjusted accordingly by adding of 1 Molar HCl (hydrochloric acid) / 1 Molar NaOH (sodium
123
hydroxide) drop wise. The change in the operational volume of the medium due to pH
124
adjustment was considered negligible. To know the direct relationship between Mn
125
solubilization and effect of pH, an uninoculated medium with pH 6 was taken as control. The
126
control as well as the experimental flasks were incubated at 37 0C.The leached liquor was
127
collected continuously at the interval of 3 days, centrifuged at 8000 rpm for 10 min and the
128
supernatant obtained was used for estimation of Mn percentage. The Mn bioleaching
129
experimentation was applicable within at most 1 % error.
130 131
2.3.2. Effect of pulp density
132
Mn solubilization by Aspergillus oryzae MSF-5 was monitored at different initial Mn
133
concentrations ranging from 2 % to 6 %. The sterilized medium was incubated at 37 0C with
134
shaking velocity at 150 rpm (Remi C-24BL) for 20 days. The effect of pulp density on Mn
135
bioleaching by pre-grown cells of MSF-5 was observed.
136 137 138
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2.3.3. Effect of carbon source
140
Fungal solubilization of Mn is extensively inclined by e- donors / carbon sources. The influence
141
of different carbon dosage like dextrose, sucrose, fructose and glucose were investigated on the
142
production of fungal biomass during Mn bioleaching. Experiments were conducted in 250 ml
143
sterilized conical flasks with 90 ml of mineral salt medium containing yeast extract - 0.5 gm/L,
144
NH4SO4 - 0.5 gm/L, MgSO4.7H2O - 0.1 gm/L, K2HPO4 - 1.5 gm/L, Nacl - 0.2 gm/L and 10 ml
145
fungal inoculum. 2 % (w/v) of each dextrose, sucrose, fructose, glucose were added separately
146
to the flasks. The pH of was adjusted to 6 by adding 1 Molar HCl or 1 Molar NaOH solution
147
drop wise. The final fungal biomass growth was measured after a period of 20 days.
148 149
3. Bioleaching of manganese
150
Bioleaching experiment was prepared in 250 ml sterilized flasks comprising 90 mL of selected
151
minimal salt media and supplemented with 2 % (w/v) of Mn ore. Sterilized conical flasks with
152
un-inoculated media served as control. Thesalt medium was autoclaved at 121 0C for 15
153
minutes and to that autoclaved medium 10 ml of fungal inoculum was added separately to the
154
above selected medium and incubated at 37 0C with a shaking velocity of 150 rpm. All the
155
experimentation, including the un-inoculated controls, was conducted in duplicates. The course
156
of bioleaching by A. oryzae in efficient recovery of Mn was measured periodically. Leached
157
liquor was collected by disposable sterilized pipettes and filtered. The final leached samples
158
were considered for final Mn concentration after a period of 20 days.
159
The percentage of Mn content after leaching was deliberated by subsequent equation:
160
Percentage (%) of Mn removal= {(ML/MS) x 100}
161
Where, ML is the sum of Mn concentrates in leached liquor in mg,
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MS is the sum of Mn in the sample before bioleaching in mg. (Mulligan et al.,2004)
163
For estimating Mn content after bioleaching, the residues of leached liquors were collected at
164
regular intervals. The collected liquors were centrifuged at 10, 000 rpm for 15 minutes and then
165
the final Mn concentration was deduced using titration method (150 ml of 0.1 Molar of EDTA
166
in burette) (Mohanty et al., 2016).
167 168
4. Microscopic analysis
169
Microscopic analysis was carried out to determine the effect of fungal species on the ore
170
residues. The presence and absence of Mn in the ore before and after bioleaching was studied
171
using SEM and EDX analysis. The fungal mycelium grown in presence and absence of 50 gm/L
172
of Mn ore in minimal salt medium was accepted as treated and untreated fungal samples
173
respectively. The pellets obtained were rinsed to about four to five times with double distilled
174
water, and then air dried for 10 minutes. The treated ore sample were examined by SEM analysis
175
and then evaluated with the control (untreated ore). The SEM images of the ore surface before
176
and after leaching by the fungal strains were observed at a magnification of 1,000X and the
177
analysis was carried out at an accelerating voltage of 50 kV. The same suspended Mn ore
178
samples were analyzed for desired and preferred elements under full scale of EDX spectrum.
179 180
5. Fungal proteins extraction
181
Isolated fungal cells were collected in eppendorf tubes and were separated from the broth media
182
at 8,000 - 10,000 rpm centrifugation for 10 minutes. The supernatant (media) was removed then
183
the remaining pellet (containing fungal mycelium) were homogenized by adding chilled (1
184
g/ml) Phosphate Buffer Saline (PBS) (General PBS concentration: 0.2 g/L potassium chloride
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(KCl) Sodium chloride (NaCl) – 8 g/L, 0.2 g/L potassium dihydrogen phosphate (KH2PO4),
186
1.15 g/L, 1L De-ionized water at pH 7.4) using sterilized micropestle.
187 188
5.1. Protein extraction and profiling by SDS-PAGE electrophoresis
189
Fungal extracted proteins ranging from 20-100 mg were loaded separately in each well of multi
190
welled acrylamide gel. Separation of protein based on their molecular weight was done by SDS-
191
polyacrylamide using a BioRad vertical electrophoresis unit. Standard protein markers were
192
also loaded and run in parallel laterally with the protein samples. The cell pellet obtained was
193
mixed with 1 ml of sample buffer (1.25 % of β mercapto-ethanol, 2.5 % of glycerol, 0.5 % of
194
Sodium Dodecyl Sulphate, 0.03 % of bromophenol blue, 15 mM TrisCl, at pH 6.8). The sample
195
buffer is incubated in a boiling water bath for 30 minutes. This was further used for protein
196
profiling using SDS-PAGE. 15 μL of the protein sample was used for SDS-PAGE holding 2.5
197
% stacking and 12.5 % of resolving gels. A 200 Kda protein marker (Sigma) was used to
198
identify the protein bands of the Mn treated as well as untreated microorganisms. Protein
199
separation was conducted under an even current of 100V. The operated gels were stained with a
200
staining solution called Coomassie Brilliant Blue R-250 (Sigma) 45 % methanol, 10 % acetic
201
acid for one hour and destained for 12 hr in 10% acetic acid and 20% methanol (v/v). Gel was
202
removed from the electrophoresis apparatus and it was stained with coomassie brilliant blue
203
stain composed of 0.25 gm coomassie brilliant blue R250 dissolved in 40 ml methanol followed
204
by addition of 10 ml glacial acetic acid and the volume was made up to100 ml. After the stain
205
was drained out from the gel, it was treated with a de staining solution. The gel was washed
206
with doubled distilled water until the gel becomes clear prior to view. Finally, the gel was
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viewed and photographed with Biorad gel doc system to capture the picture of fungal proteins
208
bands and molecular weight markers.
209 210
6. Fourier transform infrared spectroscopy (FTIR) measurements
211
FTIR spectra of untreated Mn ore, Aspergillus oryzae and Mn ore treated with fungal mycelium
212
were examined to ascertain the changes in functional groups inflicted due to the interaction of
213
ore with fungal cells. FTIR reveals various groups associated with lipid, carbohydrates and
214
proteins during microbe-mineral alteration evaluated with physiological changes. In response to
215
mineral stress, the metabolic activity of fungus alters the chemical functional groups which can
216
be visible within an absorbance range of 4000 to 400
217
interacted with Mn with shaking velocity at 150 rpm for 15 days at 37 0C in 250 mL flasks.
218
After incubation for 15 days, the leached fungal biomass was centrifuged at 7,000 rpm for 25
219
minutes (4 0C). The suspension was filtered, supernatant was discarded and then rinsed with
220
double distilled water 3 times. The treated fungal mycelium and the untreated fungus were air
221
dried and then transferred to autoclaved round bottom flask for lypholization for 5 hrs. The
222
lypholized samples were then transferred to eppendorf. Then dried potassium bromide (KBr) in
223
the ratio of 1:100 was mixed with 50 gm of samples and powdered to fine mixture. Then the
224
mixed samples were compressed to prepare a salt disc by using vacuum pressure applying 1200
225
psi for about 10 minutes. The band position of IR was presented in wave number which is
226
represented in the unit cm-1 (Table 1). The infra spectra were acquired with the help of FTIR and
227
recorded between 4000 and 400 cm-1.
228 229
10
cm-1.
FTIR analysis of fungal mycelium
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7. Chemicals and instruments used
232
Pure and analytical grade chemicals from Himedia were used in all experiments. Continuous
233
shaking condition was maintained using Incubator shaker Remi RS-24BL. FTIR analysis was
234
conducted by using model 4100, JASCO.
235 236
3. Results
237
3.1. Sample characterization
238
Low grade ore sample was studied for their physicochemical properties and was found to have a
239
pH of 8.67, conductivity of 3.62 S/m and particle dimensions ranging from 8 to 18 mm. The
240
low grade ore sample reported a TSS concentration of 6.59 g/L and TDS concentration of 1.03
241
g/L. As obtained in our previous paper (Mohanty et al., 2016) the content of Mn was valued to
242
about 0.21 g/L i.e 21 %.
243
3.2. Fungal species characterization
244
Out of all fungal species isolated from mining site, Aspergillus oryzae MSF-5 (KP309810) was
245
selected for this study due to its highest tolerance to Mn (Mohanty et al., 2016). The growth
246
percentage of strain MSF-5 in Mn added culture medium was also more rapid in comparison to
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the other isolated fungal strains. Rapid growth of this mycelium species is seen typically after 5-
248
7 days of inoculation.Aspergillus oryzae has been well reported as a fungal species associated
249
with Mn bioleaching (Amin et al., 2013; Dwivedi et al., 2010) recovered from resources such as
250
deep-sea sediments, sedimentary rock samples.
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3.2. Optimization of parameters
255
3.2.1. Effects of pH
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Mn bioleaching by Aspergillus oryzae was studied in the pH range 2-8 in 2 % Mn
257
concentration. An increase in biomass growth was observed at pH ~5-6 (Fig.1) and the fungal
258
strain showed highest percentage of Mn solubilization at pH ~6.The growth of mycelium is
259
observed with a varied range of pH 5-6 (Fig. 1). It is clear that bioleaching is directly related to
260
the biomass growth. At optimum pH, growth is maximum so metabolic activity is maximum
261
which leads to better Mn leaching. (Acharya et al., 2003) reported elevation in Mn removal by
262
Aspergillus niger with decrease of pH. (Gholami et al., 2010) suggested high metal binding
263
activity with increase in pH due to decrease in proton concentration, as they also compete for
264
the binding sites. Most of the reducing microorganisms grow optimally at pH 5 to 6.5 because
265
primary acidic situations produce tough reducing atmosphere and the microbes can uphold
266
homeostasis at pH 6.5 avoiding intracellular damage (Acharya et al., 2003).
267
The highest observable growth in addition to Mn reduction was calculated at pH 3 with
268
highest efficiency in 4th day of leaching. However, various forms of Mn are soluble with a
269
varied range of pH and are observed to be usually mobile in water and soil environs (Li et al.,
270
2013). According to (Roane and Pepper, 1999), pH of the system may have considerable
271
solubilizing effect on heavy metal and henceforward their metal bioavailability. As reported by
272
(Mehta et al., 2010) the effective impact of microbial inhabitants by distressing their
273
development, external feature, bio-chemical processes eventually observed subsequent decrease
274
in their pH and other extracellular activities (Roane and Pepper, 1999).
275
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3.3.2. Effect of pulp density
278
The effect of initial concentration on Mn solubilization by Aspergillus oryzae was examined
279
using pulp density ranging from 2 % to 6 %. Substantial Mn solubilization occurred over the
280
entire Mn concentration range studied (Fig. 2). Maximum biomass growth of fungal mycelium
281
was observed at lower Mn initial concentrations of 2 % after 20 days. At pulp density 2 % and
282
6%, 2.119 gm/L and 0.533 gm/L of fungal biomass growth was recorded within 20 days of
283
incubation, respectively showing its relevant growth rate. The resultant toxicity of Mn acquired
284
due to the mutagenic effect on various microbes have been earlier described with concentrations
285
at 5- 10 mg/L of Mn compounds. The above concentration shows an inhibitory effect to almost
286
all fungal mycelium in cultured medium (Amin et al., 2014).
287 288
3.2.3. Effect of carbon sources
289
Microbial solubilization by Aspergillus oryzae of Mn has a significant correlation to carbon
290
dosage. Effect of several carbon sources like sucrose, fructose, dextrose and glucose in Mn
291
bioleaching was investigated. Maximum bleaching was observed in case of dextrose and hence
292
it was chosen for carrying out further experiments (Fig. 3). Maximum growth of fungal biomass
293
weight was obtained with dextrose after 7 days, serving as the effective carbon source for Mn
294
solubilization. It is well known that toxic Mn(II) was transformed to non-toxic Mn(VI) by a
295
microbial mechanism where the electrons are transferred to Mn(II) during bioleaching (Liu et
296
al., 2008). Hence, we can conclude that the carbon source (dextrose) act as electron donor in
297
this method and having the maximum capability of reducing activity of Mn.Thus, microbes
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result with a highest growth with the respective carbon dosage by addition of different carbon
299
sources (Li et al., 2008).
300
4. Bioleaching study
301
Mn bioleaching capability by fungal mycelium is significantly raised as the strain adapts itself
302
to tolerate high concentration of Mn (Das et al., 2011). The experimental results for bioleaching
303
showed 79 % of Mn recovery by Aspergillus oryzae (KP309810) within 20 days of leaching
304
study. The process of bio-oxidation and bio-reduction by bioleaching of Mn by various fungi
305
involves a significant method for the development of insoluble Mn (III, IV) in the expected
306
surroundings as depicted by (Gadd, 2010). Metal bioleaching by heterotrophic fungal strains
307
usually comprise an indirect method with macrobiotic generation of natural acids (gluconic
308
acid, lactic acid, oxalic acid, citric acid), some assimilated amino acids, and other simpler
309
metabolites (Das et al., 2012). As projected by (Behera et al., 2015), the intercellular
310
mechanism of Aspergillus sp. involves the conversion of sugar compounds (sucrose, glucose)
311
into different organic acids leading in decreasing the pH of the medium. The generated
312
metabolic products generally have the diplomatic consequence of dissolving insoluble metal
313
complexes from minerals by reducing the level of pH and rising concentration of soluble metals
314
by complexing (Bahh et al., 2013).
315
However various group of fungi are being involved in boosting oxidation of Mn which is
316
probably due to enzymatic and non-enzymatic interaction with the fungal metabolic product.
317
(Chen et al., 2014) accounted that Mn ion is measured as the most significant metal for cellular
318
metabolic activity. Thus, the existence of Mn ions accelerates the activation of enzymatic action
319
over elevation of oxalate compound. The cofactor of Mn is positioned in the cytoplasmic area of
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Asperigillus niger, afterward it metabolizes the alteration of oxalo-acetate to acetate and oxalate
321
(Han et al., 2013).
322 323
5. Microscopic analysis
324
The SEM micrographs of Mn ore surface leached and unleached with fungal mycelium cells
325
are depicted in Fig. 4. Morphology observed after SEM analysis reveals that the surface of Mn
326
ore is smoother than that of ore surface exposed to fungal leaching. However, SEM image of
327
1000 X ore particles vary largely in arrangement of crystals viewed in the given figure. In the
328
other hand, a big sum of deposits in a pattern of comparative free porous, rough, coagulated
329
precipitates are observed on the revealed surface of leached sample ore treated with fungus after
330
20 days of bioleaching (Das et al., 2015a). It is of course demonstrated that the ore treated with
331
fungus views a fraction of bulky quartz particles. Chemical and metallic constituents were
332
determined by using an EDX spectrometer in (Fig. 4 C, D, E). Micrographs illustrate the shape
333
of the ore particles and results suggest that the ore was a heterogeneous material containing a
334
large particle size distribution (Fig. 4 A, B). Analysis of the particles with EDX indicates that
335
Mn along iron, aluminum and silicon are present in the ore sample. Similar studies on
336
morphological characters and elemental chemical analysis of the ore from chromite mines (Das
337
and Mishra, 2010; Xin et al., 2010; Zhang et al., 2008).
338 339
6. Estimation of total protein and protein profiling
340
The total protein content of fungal strains Aspergillus oryzae was found to be 28.38 g/l. The
341
visible molecular mass of fungal strains after SDS-PAGE analysis produced a multiple band of
342
protein. It is evident from the figure that the bands obtained with separated protein according to
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their molecular weight as 116, 97, 66, 55, 36 and 14.2 kilo Dalton (kDa) protein bands were
344
observed with more thickness in presence of Mn. Aspergillus oryzae contains 20 protein bands
345
with the minimum band from 14.2 to 97 KDa (Fig. 5). The alteration of protein bands of fungal
346
species after treatment with Mn ore may be obtained due to bioleaching activity beyond the
347
optimum temperatures or attributed due to denaturation of the proteins involved in the oxidizing
348
system of the fungus caused by the increase in the rate of thermal death of the microorganisms
349
(Durve et al., 2013). The changes in the conformation of protein patterns is basically because
350
Mn ion may alter the biological membranes, extracellular and intracellular enzyme specified
351
mutation and damage the cellular and structural variabilities (Roane and Pepper, 1999).
352
In observing the effect of the toxicity of metal ions onto adequate exposure of metal, the
353
sensitivity of microbe-metal interaction will outcome with instant death of the cellular
354
components. Due to their damage in the essential components of cell, inclined changes in the
355
outline of population dimensions are acquired (Wyatt, 2015).
356 357
7. Fourier transform infrared spectroscopy (FTIR) analysis
358
FTIR analysis was carried out using model FTIR-4100, JASCO instrument. The IR spectrum of
359
Aspergillus oryzae mycelium cells without Mn and IR spectrum of all the three fungus with Mn
360
ore treated was documented in the spectrum ranging (4000 and 400 cm-1) (Table 1).
361 362
7.1. Characterization of MnOx
363
Collected low grade Mn ore samples were grounded and powdered finely and analyzed for
364
FTIR analysis and their peaks were obtained at 1099.80 cm-1, 582.40 cm-1, 476.33 cm-1, 417.51 cm-1
16
ACCEPTED MANUSCRIPT
cm-1
and 476.33
cm-1
365
(Fig. 6 A). The acquired peaks observed at 582.40
366
stretching and CH2 vibrations as described by (Buciuman et al., 2005).
is recognized as MnOx
367 368
7.2. FTIR Spectral characterization of fungal cells (Mn+/Mn-)
369
Infra-red (IR) spectrum of dry biomass cells of Aspergillus oryzae (Fig. 6 B) and Mn ore treated
370
Aspergillus oryzae were obtained in (Fig. 6 C). The observed major variation of peaks obtained
371
by untreated at 1631.48 cm-1, 1089.58 cm-1, 775.24 cm-1, 688.46 cm-1and 464.76 cm-1 (Table 2). The
372
above obtained peaks generally understand the probable metal-cell interaction that codes for
373
functional groups like C=C group (Amide I), C-N stretching (Aliphatic amines), N-H wag
374
(primary (10) and secondary (20) amines), -C=C- stretching (alkynes) and CH2 vibrations
375
(Polysaccharides) respectively (Table 3). While on the other hand the spectrum of Mn treated
376
Aspergillus oryzae shifted the peaks to 3384.46
377
1377.89
378
corresponding to phenols, alcohols compounds, C=C group (alkanes), -C=C stretch (amide I), -
379
C-O stretch (alcohols, carboxylic acid,). The changes in C-N stretching compare with aliphatic
380
amines, while CH2 vibration correlates to polysaccharides (Parikh and Chorover, 2005).
cm-1,
1249.65
cm-1
and 569.86
cm-1.
cm-1,
2926.45
cm-1
1650.77
cm-1,
1545.67
cm-1,
These peaks represent -OH group C-H stretching
381
Formation of newer carbon (C) and nitrogen (N) bonds in Mn treated fungal species is
382
believed to change the conformation of protein patterns. The change in corresponding peaks
383
suggests conformational alteration in protein amide patterns due to fungal bioleaching that may
384
be acquired because of enzymatic activity (Roy et al., 1994). Biomining remediation of Mn (II),
385
utilizing fungal mycelium has been observed in biologically intracellular metal accumulation
386
converting the functional groups using direct bio-transformation with functional metabolites.
387
(Kalinowska et al., 2014) carried out the FTIR spectroscopic analysis providing instantaneous
17
ACCEPTED MANUSCRIPT
388
molecular level metabolism through their organic and inorganic dynamic interaction with the
389
metal surface. FTIR also reveals the study of various groups associated with lipid,
390
carbohydrates and proteins during microbe-mineral alteration evaluated with physiological
391
changes.
392
8. Possible mechanism for variation in protein expression
393
In most instances, some fungal species have also been reported to promote Mn oxidation due to
394
interaction with metabolites i.e bio-generated acid generated from fungal cell component. The
395
capability of indigenous microbes to tolerate Mn heavy metals is sustained by different basic
396
mechanisms which comprise of intracellular enzymatic alteration, bioreduction in sensitivity of
397
cellular surfaces, metal and cellular elongation. On the other hand, oxidation resulted may be
398
due to extracellular presence of Mn(II) peroxidase. Other additional enzymes for altering the
399
expression of protein patterns such as cellobiose dehydrogenase (CDH), may predominantly
400
participate in reduction and oxidation cycling of Mn (Nealson et al., 2006) confirmed that the
401
enzyme cellobiose dehydrogenase can decrease the concentration of insoluble MnO2 and
402
generate Mn(II), the substrate for MnP. Several investigators (Nealson, 2006; Vrind et al., 1986)
403
have examined that Mn(II) oxidizing microorganisms can transfer Mn oxides as an e- acceptor
404
for the continued existence under anaerobic or microaerophili conditions. The enzyme
405
manganese peroxidases secreted by fungus are utilized to catalyze the bio-transformation of
406
Mn(III) compounds that can also consequently oxidize the bioleaching activity and altering the
407
protein patterns ultimately (Das et al., 2015). We can thus conform that different fungal strains
408
participates in enhanced mechanism of Mn biomass interaction will come forward as important
409
mineral large scale remediation due to their higher tolerance of acute conditions of
410
contaminated mining sites.
18
ACCEPTED MANUSCRIPT
411 412
9. Conclusions
413
Bioleaching is an economic process of recovering minerals from low to medium grade ores and
414
waste residues using microorganisms. As there is no substitute for Mn in its major application,
415
the demand for Mn is ever increasing due to rapid industrialization and gradual depletion of
416
high grade ore and the rising insufficiency of natural deposits. The conventional pyro-
417
metallurgical methods used for extraction of Mn from are very expensive, requires lots of
418
energy and additionally causes environmental pollution problems Bioleaching is considered as a
419
persuading way to solve these problems. In the current investigation leaching of Mn from
420
mining waste ore using native isolated fungi Aspergillus oryzae was investigated. Influence of
421
operational conditions on bioleaching of Mn ore with Aspergillus sp. were examined for
422
duration of 20 days. The optimal conditions for enhanced recovery of Mn (79 %) were observed
423
to be at temperature 37 0C, pH 6 and pulp density 2 % (w/v). Increasing metal concentrations
424
seem to be the reason behind the shifting in proteomic data of the fungal cells during the
425
bioleaching procedure. Mn and protein interaction results in the alteration of amide
426
configuration and may also modify the conformational changes in functional groups due to Mn
427
solubilization.
428
It is very clear that proteins play a significant role in mineral-microbe interphase with ores.
429
The present study of deviation of protein expression and conformation had been valuable in
430
supervising the modification that occurs in the fungal cells during bioleaching. Study of
431
advance contact mechanisms are needed to recognize specific proteins concerned in the
432
bioleaching procedure so that the information can pave a way for improved biorecovery of
433
metals from their ores.
19
ACCEPTED MANUSCRIPT
434 435
Acknowledgment
436
Authors express their thanks to the (DBT) Department of Biotechnology Technology,
437
Government of India (BT/PR7454/BCE/8/949/2012), Department of Science and Technology
438
(DST) (SP/YO/031/2016) and Ministry of Mines Government of India (07/46SSAG/CAT) for
439
granting projection in manganese biomining and recycling studies.
440
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581 582
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Fig. 1. Effect of pH on Mn solubilization by A. oryzae
1
ACCEPTED MANUSCRIPT
Fig. 2. Effect of pulp density on Mn solubilization by A. oryzae
2
ACCEPTED MANUSCRIPT
Fig. 3. Effect of carbon sources on Mn solubilization by A. oryzae.
3
ACCEPTED MANUSCRIPT
Fig. 4. Microscopic analysis of treated and untreated ore samples through SEM/EDX (A) SEM analysis of Mn ore samples (B) SEM analysis of fungal treated ore sample with magnification of 1000 X (C) photomicrograph of EDX with electron image at 100um (D) EDX images of Mn ore sample with elevated peaks of elements likeMn ,
4
ACCEPTED MANUSCRIPT
Fig. 5. Conformational protein patterns by SDS PAGE of Mn ore treated and untreated Asperigillus orzyae (MSF 7)
5
ACCEPTED MANUSCRIPT
Fig. 6. Fourier transform infrared spectroscopy (FTIR) peaks in cm-1 (A) IR spectra of low grade Mn ore (B) IR peaks of Aspergillus oryzae (C) IR peaks of Mn ore treated Aspergillus oryzae.
6
ACCEPTED MANUSCRIPT
Fig. 5. Conformational protein patterns by SDS PAGE of Mn ore treated and untreated Asperigillus orzyae (MSF 7)
Table 1: FT-IR analyzed wave number frequencies in cm-1 and their assigned groups
Frequency (cm-1)
ACCEPTED MANUSCRIPT Functional group Assigned
Below 500 cm -1
C-C bending vibrations occur at very low frequencies.
750-200 cm -1
MnOx stretching, bending, and wagging vibrations
650-450 cm -1
CH2 vibrations of polysaccharides
1200- 800 cm -1
C-C stretching vibrations are weak and in this region.
1439-1399 cm -1
C-H bending vibrations methyl groups.
1340- 1400 cm -1
Overlapping doublets for t-butyl and isopropyl group
3000-2840 cm -1
C-H stretching in the alkanes.
2962 & 2872 cm -1
Saturated hydrocarbons containing methyl groups.
1
Table 2 IR spectra peaks of Mn ore sample
Peaks values
Bond assigned MANUSCRIPT Assignment ACCEPTED
Functional group
(cm-1) 1099.80
C-N stretching
(C-N)ar
Aliphatic amines
582.40
CH2 vibrations
(CH2)ar
Polysaccharides
476.33
MnOx stretching, bending, and
(MnOx)
a-Mn2O3
(MnOx)
b-MnO2
wagging vibrations 417.51
MnOx stretching, bending, and wagging vibrations
Table 3. IR peaks in cm-1of Aspergillusoryzaeand Mn ore treated Aspergillusoryzae Aspergillusoryzae Peaks values (cm-1)
Bond
Assignment
1631.48
C=C group
v(C═O)
1089.58
C-N stretching N-H wag
(C-N)ar
775.24
688.46 540.94 464.76
Mn ore treated Aspergillusoryzae Functional group
Peaks values (cm-1)
Bond
Amide I: 3384.46 -OH group ACCEPTED MANUSCRIPT C═O, C═ N, N═ H Aliphatic amines 2926.45 C-H stretching
Assignment
Functional groups
v(OH)ar
Phenols, alcohols
(CH)ar
Alkanes
(N-H)ar
Primary (1O) Secondary(2O) amines
1650.77
C=C group
v(C═O)
Amide I: C═O, C═ N, N═ H
-C=Cstretch CH2 vibrations
v(C═C)
alkynes
1545.67
-C=C stretch
v(C═C)-c=c–
(CH2)ar
Polysaccharides
1377.89
-C-O stretch
v(COO−)
CH2 vibrations
(CH2)ar
Polysaccharides
1249.65
C-N stretching
(C-N)ar
Amide I: N═H, C═ N Alcohols, Carboxylic acid, esters, ethers Aliphatic amines
569.86
CH2 vibrations
(CH2)ar
Polysaccharides
a,c
Sansuta Mohanty, Shreya Ghosh, Sanghamitra Nayak, Alok Prasad Das PII:
S0045-6535(16)31879-3
DOI:
10.1016/j.chemosphere.2016.12.136
Reference:
CHEM 18592
To appear in:
Chemosphere
Received Date:
18 October 2016
Revised Date:
23 December 2016
Accepted Date:
27 December 2016
Please cite this article as: Sansuta Mohanty, Shreya Ghosh, Sanghamitra Nayak, Alok Prasad Das, Bioleaching of manganese by Aspergillus oryzae isolated from mining deposits, Chemosphere (2016), doi: 10.1016/j.chemosphere.2016.12.136
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ACCEPTED MANUSCRIPT Highlights 1. It elucidates the mechanism of manganese bioleaching by Aspergillus oryzae and the comprehensive factors that emphasize the selection of manganese recovery technique. 2. Includes the understanding of alteration of fungal protein expression in response to Mn bioleaching.
3. Elemental and mineralogical studies of the ore sample were carried out via microscopic analysis, SEM, EDX, XRD and FITR to access the basic arrangement of preferred elements and functional groups.
ACCEPTED MANUSCRIPT
1
Bioleaching of manganese by Aspergillus oryzae isolated from mining deposits
2
Sansuta Mohantya, Shreya Ghosha, Sanghamitra Nayak a and Alok Prasad Dasb*
3 4 5
aBioengineering
& Biomineral Processing Laboratory, Centre of Biotechnology,
Siksha O Anusandhan University, Khandagiri Square, Bhubaneswar, India bDepartment
of Chemical and Polymer Engineering, Tripura University (A Central
6
University), Suryamaninagar, Tripura
7
Email: [email protected]
8 9 10 11 12 13 14 15 16
Corresponding Author: Dr. Alok Prasad Das*
17
Department of Chemical and Polymer Engineering, Tripura University (A Central University),
18
Suryamaninagar, Tripura.
19
Email: [email protected]
20
Phone: +919178581814
21 22 23
1
ACCEPTED MANUSCRIPT
24 25
Abstract
26
A comprehensive study on fungus assisted bioleaching of manganese (Mn) was carried out to
27
demonstrate Mn solubilization of collected low grade ore from mining deposits of Sanindipur,
28
Odisha, India. A native fungal strain MSF 5 was isolated and identified as Aspergillus oryzae by
29
Inter Transcribed Spacer (ITS) sequencing. The identified strain revealed an elevated tolerance
30
ability to Mn under varying optimizing conditions like initial pH (2, 3, 4, 5, 6, 7), carbon sources
31
(dextrose, sucrose, fructose and glucose) and pulp density (2 %, 3 %, 4 %, 5 % and 6 %).
32
Bioleaching studies carried out under optimized conditions of 2% pulp density of Mn ore at pH
33
6, temperature 37 0C and carbon dosage (dextrose) resulted with 79 % Mn recovery from the ore
34
sample within 20 days. SEM-EDX characterization of the ore sample and leach residue was
35
carried out and the micrographs demonstrated porous and coagulated precipitates scattered
36
across the matrix. The corresponding approach of FTIR analysis regulating the Mn oxide
37
formation shows a distinctive peak of mycelium cells with and without treated Mn, resulting
38
with generalized vibrations like MnOx stretching and CH2 stretch. Thus, our investigation
39
endeavors’ the considerate possible mechanism involved in fungal surface cells onto Mn ore
40
illustrating an alteration in cellular Mn interaction.
41 42
Keywords: Bioleaching, Aspergillus oryzae, Optimization, Protein profiling, SEM, FTIR
43 44 45 46
2
ACCEPTED MANUSCRIPT
47 48
1. Introduction
49
Indiscriminate exploration of heavy metal with major environmental setbacks is one of the
50
major challenges of widespread mining (Bing et al., 2012; Ghosh et al., 2015). The liberation
51
of toxic heavy metals into the atmosphere through industrial effluents is the foremost concern.
52
Mn mining operations deeply provide a profitable advancement although at the same time it
53
depreciates the environment through metal pollutants from mining. Widespread mining and
54
random disposal of mining wastes are principally responsible for environmental contamination.
55
The estimated amount of solid waste generated is about 7-8 million tones in the form of
56
disposed minerals, waste sources and lean grade ore to ensuing in ecological pollution (Akcil et
57
al., 2015). As manifested from the current study, Mn contaminated soils cover common metal
58
contaminants in low and high concentration of different types of metals as pollutants.
59
Integration of heavy metal contaminants in environment generally leads to variations in
60
biochemical speciation. However, it is a significant aspect to work on the metal bioavailability
61
onto the toxicity of a heavy metal that are largely depended on the influences of metal ions.
62
(Roane and Kellogg, 1996).
63
The mineral bioleaching ability of Mn solubilizing microorganisms illustrates an essential
64
part for further exploration of this metal from various sources (Ghosh et al., 2015). Recent study
65
reported that (Vera et al., 2013) examined that the oxidation of Mn may involve the alteration of
66
various proteins for regulation of Mn solubilization focusing the induction of insoluble Mn
67
(Myers et al., 2003) oxides forming soluble Mn(III) complex. However, the response of Mn
68
solubilization occurs naturally through enzymatic activity involving enzymes like multicopper
69
oxidase (MCO), cellobiose dehydrogenase (CDH), Manganese peroxidase (MnP), confirming
3
ACCEPTED MANUSCRIPT
70
the biotransformation of protein patterns of different fungal strains (Hakala et al., 2006;
71
Podorozhko et al., 2008).
72
On considering the effective Mn solubilization by fungal mycelium, our present study aims
73
(1) in optimizing different parameters like initial pH (2, 3, 4, 5, 6, 7), carbon sources (dextrose,
74
sucrose, fructose and glucose) and pulp density (2%, 3%, 4%, 5% and 6%), (2) in determining
75
the Mn leaching efficiency of the isolated fungal strain Aspergillus oryzae MSF-5 under
76
optimized conditions, (3) in ascertaining the changes in the ore composition through FTIR
77
analysis. Thus, our research also endeavors the understanding of alteration of fungal protein
78
expression in response to Mn bioleaching.
79
2. Material and methods
80
2.1 Mineral sample and culture conditions
81
The lean grade Mn ore samples were collected from Sanindipur mines from Barbil of
82
Kendhujhar district, Odisha, India. The samples were finely crushed and grounded, then sieved
83
in various size fractions for leaching experiments. The crushed samples were subjected to acid
84
digestion and examined for estimating the percentage of Mn content present in the ore.
85
Mineralogical analysis of the composed Mn ore sample was examined to access the quality
86
grade. The physico-chemical parameters of the ore sample like size of the particle, TDS, TDS,
87
conductivity, pH was examined. Elemental and mineralogical studies of the ore sample were
88
carried out via microscopic analysis, SEM, EDX (JEOL JSM-6330F), XRD analysis. FITR was
89
used to access the basic arrangement of functional groups.
90 91 92
4
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93
2.2. Fungal strain and medium concentrations
94
A fungal species capable of tolerating Mn in high concentration was isolated from the ore
95
assembled from Mn dumping areas of Sanindipur mines. Based on biochemical analysis and
96
ITS
97
GGAAGTAAAAGTCGTAACAAGG-3’) and ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’)
98
the isolated fungal species was classified as Aspergillus oryzae MSF 5 (with accession number
99
KP309810). Initial culturing of the fungal strain was done on potato dextrose agar (PDA) plates
100
at 37 0C until mycelium was observed covering most of the plate surface. Fungal colonies
101
obtained from mining low grade ore were maintained onto an optimized medium i.e. minimal
102
salt medium (MSM) with concentrations like 0.5 % (w/v) Mn: yeast extract - 0.5 g/L, Dextrose
103
- 10,Ammonium sulphate (NH4SO4) - 0.5 g/L, Magnesium sulphate (MgSO4.7H2O) - 0.1 g/L,
104
Dipotassium hydrogen phosphate (K2HPO4) - 1.5 g/L, Sodium chloride (Nacl) - 0.2 g/L, Agar –
105
2 %, pH adjusted to 6.8 as discussed in our previous paper (Mohanty et al., 2016).
(internal
transcribed
spacer)
sequencing
using
primers
ITS-5
(5’-
106 107
2.3. Optimization of Mn bioleaching under different parameters
108
As Mn bioleaching is statistically altered by different parameters, the solubilization of Mn by
109
fungal strains was optimized under several parameters like Mn concentration, pH and sugar
110
(carbon dosage) to regulate the conditions for effective Mn bioleaching via fungal strains. The
111
effect of parameters like pH (2, 3, 4, 5, 6, 7), initial Mn concentration (50, 100, 200, 500 mg/L)
112
and carbon sources (dextrose, sucrose, fructose and glucose) on Mn bioleaching were
113
investigated for a period of 21 days. All the reduction experiments were carried in MSM
114
containing Dextrose-10.0 g/L, yeast extract-0.5 g/L, Ammonium sulphate (NH4SO4)- 0.5 g/L,
115
Magnesium sulphate MgSO4.7H2O - 0.1 g/L, Sodium chloride (Nacl) - 0.2 g/L, Dipotassium
5
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116
hydrogen phosphate (K2HPO4) - 1.5 g/L (Pradhan et al., 2005), pH adjusted to 6 with 10 ml of
117
fungal inoculum of A. oryzae and 2 % (w/v) of Mn concentration at pH 6. The error bars were
118
put on the graph obtained by the standard deviation.
119 120
2.3.1. Effects of pH
121
The significant effect of pH on Mn bleaching was studied in the range of pH 2 to 7. The pH was
122
adjusted accordingly by adding of 1 Molar HCl (hydrochloric acid) / 1 Molar NaOH (sodium
123
hydroxide) drop wise. The change in the operational volume of the medium due to pH
124
adjustment was considered negligible. To know the direct relationship between Mn
125
solubilization and effect of pH, an uninoculated medium with pH 6 was taken as control. The
126
control as well as the experimental flasks were incubated at 37 0C.The leached liquor was
127
collected continuously at the interval of 3 days, centrifuged at 8000 rpm for 10 min and the
128
supernatant obtained was used for estimation of Mn percentage. The Mn bioleaching
129
experimentation was applicable within at most 1 % error.
130 131
2.3.2. Effect of pulp density
132
Mn solubilization by Aspergillus oryzae MSF-5 was monitored at different initial Mn
133
concentrations ranging from 2 % to 6 %. The sterilized medium was incubated at 37 0C with
134
shaking velocity at 150 rpm (Remi C-24BL) for 20 days. The effect of pulp density on Mn
135
bioleaching by pre-grown cells of MSF-5 was observed.
136 137 138
6
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139
2.3.3. Effect of carbon source
140
Fungal solubilization of Mn is extensively inclined by e- donors / carbon sources. The influence
141
of different carbon dosage like dextrose, sucrose, fructose and glucose were investigated on the
142
production of fungal biomass during Mn bioleaching. Experiments were conducted in 250 ml
143
sterilized conical flasks with 90 ml of mineral salt medium containing yeast extract - 0.5 gm/L,
144
NH4SO4 - 0.5 gm/L, MgSO4.7H2O - 0.1 gm/L, K2HPO4 - 1.5 gm/L, Nacl - 0.2 gm/L and 10 ml
145
fungal inoculum. 2 % (w/v) of each dextrose, sucrose, fructose, glucose were added separately
146
to the flasks. The pH of was adjusted to 6 by adding 1 Molar HCl or 1 Molar NaOH solution
147
drop wise. The final fungal biomass growth was measured after a period of 20 days.
148 149
3. Bioleaching of manganese
150
Bioleaching experiment was prepared in 250 ml sterilized flasks comprising 90 mL of selected
151
minimal salt media and supplemented with 2 % (w/v) of Mn ore. Sterilized conical flasks with
152
un-inoculated media served as control. Thesalt medium was autoclaved at 121 0C for 15
153
minutes and to that autoclaved medium 10 ml of fungal inoculum was added separately to the
154
above selected medium and incubated at 37 0C with a shaking velocity of 150 rpm. All the
155
experimentation, including the un-inoculated controls, was conducted in duplicates. The course
156
of bioleaching by A. oryzae in efficient recovery of Mn was measured periodically. Leached
157
liquor was collected by disposable sterilized pipettes and filtered. The final leached samples
158
were considered for final Mn concentration after a period of 20 days.
159
The percentage of Mn content after leaching was deliberated by subsequent equation:
160
Percentage (%) of Mn removal= {(ML/MS) x 100}
161
Where, ML is the sum of Mn concentrates in leached liquor in mg,
7
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162
MS is the sum of Mn in the sample before bioleaching in mg. (Mulligan et al.,2004)
163
For estimating Mn content after bioleaching, the residues of leached liquors were collected at
164
regular intervals. The collected liquors were centrifuged at 10, 000 rpm for 15 minutes and then
165
the final Mn concentration was deduced using titration method (150 ml of 0.1 Molar of EDTA
166
in burette) (Mohanty et al., 2016).
167 168
4. Microscopic analysis
169
Microscopic analysis was carried out to determine the effect of fungal species on the ore
170
residues. The presence and absence of Mn in the ore before and after bioleaching was studied
171
using SEM and EDX analysis. The fungal mycelium grown in presence and absence of 50 gm/L
172
of Mn ore in minimal salt medium was accepted as treated and untreated fungal samples
173
respectively. The pellets obtained were rinsed to about four to five times with double distilled
174
water, and then air dried for 10 minutes. The treated ore sample were examined by SEM analysis
175
and then evaluated with the control (untreated ore). The SEM images of the ore surface before
176
and after leaching by the fungal strains were observed at a magnification of 1,000X and the
177
analysis was carried out at an accelerating voltage of 50 kV. The same suspended Mn ore
178
samples were analyzed for desired and preferred elements under full scale of EDX spectrum.
179 180
5. Fungal proteins extraction
181
Isolated fungal cells were collected in eppendorf tubes and were separated from the broth media
182
at 8,000 - 10,000 rpm centrifugation for 10 minutes. The supernatant (media) was removed then
183
the remaining pellet (containing fungal mycelium) were homogenized by adding chilled (1
184
g/ml) Phosphate Buffer Saline (PBS) (General PBS concentration: 0.2 g/L potassium chloride
8
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185
(KCl) Sodium chloride (NaCl) – 8 g/L, 0.2 g/L potassium dihydrogen phosphate (KH2PO4),
186
1.15 g/L, 1L De-ionized water at pH 7.4) using sterilized micropestle.
187 188
5.1. Protein extraction and profiling by SDS-PAGE electrophoresis
189
Fungal extracted proteins ranging from 20-100 mg were loaded separately in each well of multi
190
welled acrylamide gel. Separation of protein based on their molecular weight was done by SDS-
191
polyacrylamide using a BioRad vertical electrophoresis unit. Standard protein markers were
192
also loaded and run in parallel laterally with the protein samples. The cell pellet obtained was
193
mixed with 1 ml of sample buffer (1.25 % of β mercapto-ethanol, 2.5 % of glycerol, 0.5 % of
194
Sodium Dodecyl Sulphate, 0.03 % of bromophenol blue, 15 mM TrisCl, at pH 6.8). The sample
195
buffer is incubated in a boiling water bath for 30 minutes. This was further used for protein
196
profiling using SDS-PAGE. 15 μL of the protein sample was used for SDS-PAGE holding 2.5
197
% stacking and 12.5 % of resolving gels. A 200 Kda protein marker (Sigma) was used to
198
identify the protein bands of the Mn treated as well as untreated microorganisms. Protein
199
separation was conducted under an even current of 100V. The operated gels were stained with a
200
staining solution called Coomassie Brilliant Blue R-250 (Sigma) 45 % methanol, 10 % acetic
201
acid for one hour and destained for 12 hr in 10% acetic acid and 20% methanol (v/v). Gel was
202
removed from the electrophoresis apparatus and it was stained with coomassie brilliant blue
203
stain composed of 0.25 gm coomassie brilliant blue R250 dissolved in 40 ml methanol followed
204
by addition of 10 ml glacial acetic acid and the volume was made up to100 ml. After the stain
205
was drained out from the gel, it was treated with a de staining solution. The gel was washed
206
with doubled distilled water until the gel becomes clear prior to view. Finally, the gel was
9
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207
viewed and photographed with Biorad gel doc system to capture the picture of fungal proteins
208
bands and molecular weight markers.
209 210
6. Fourier transform infrared spectroscopy (FTIR) measurements
211
FTIR spectra of untreated Mn ore, Aspergillus oryzae and Mn ore treated with fungal mycelium
212
were examined to ascertain the changes in functional groups inflicted due to the interaction of
213
ore with fungal cells. FTIR reveals various groups associated with lipid, carbohydrates and
214
proteins during microbe-mineral alteration evaluated with physiological changes. In response to
215
mineral stress, the metabolic activity of fungus alters the chemical functional groups which can
216
be visible within an absorbance range of 4000 to 400
217
interacted with Mn with shaking velocity at 150 rpm for 15 days at 37 0C in 250 mL flasks.
218
After incubation for 15 days, the leached fungal biomass was centrifuged at 7,000 rpm for 25
219
minutes (4 0C). The suspension was filtered, supernatant was discarded and then rinsed with
220
double distilled water 3 times. The treated fungal mycelium and the untreated fungus were air
221
dried and then transferred to autoclaved round bottom flask for lypholization for 5 hrs. The
222
lypholized samples were then transferred to eppendorf. Then dried potassium bromide (KBr) in
223
the ratio of 1:100 was mixed with 50 gm of samples and powdered to fine mixture. Then the
224
mixed samples were compressed to prepare a salt disc by using vacuum pressure applying 1200
225
psi for about 10 minutes. The band position of IR was presented in wave number which is
226
represented in the unit cm-1 (Table 1). The infra spectra were acquired with the help of FTIR and
227
recorded between 4000 and 400 cm-1.
228 229
10
cm-1.
FTIR analysis of fungal mycelium
ACCEPTED MANUSCRIPT
230 231
7. Chemicals and instruments used
232
Pure and analytical grade chemicals from Himedia were used in all experiments. Continuous
233
shaking condition was maintained using Incubator shaker Remi RS-24BL. FTIR analysis was
234
conducted by using model 4100, JASCO.
235 236
3. Results
237
3.1. Sample characterization
238
Low grade ore sample was studied for their physicochemical properties and was found to have a
239
pH of 8.67, conductivity of 3.62 S/m and particle dimensions ranging from 8 to 18 mm. The
240
low grade ore sample reported a TSS concentration of 6.59 g/L and TDS concentration of 1.03
241
g/L. As obtained in our previous paper (Mohanty et al., 2016) the content of Mn was valued to
242
about 0.21 g/L i.e 21 %.
243
3.2. Fungal species characterization
244
Out of all fungal species isolated from mining site, Aspergillus oryzae MSF-5 (KP309810) was
245
selected for this study due to its highest tolerance to Mn (Mohanty et al., 2016). The growth
246
percentage of strain MSF-5 in Mn added culture medium was also more rapid in comparison to
247
the other isolated fungal strains. Rapid growth of this mycelium species is seen typically after 5-
248
7 days of inoculation.Aspergillus oryzae has been well reported as a fungal species associated
249
with Mn bioleaching (Amin et al., 2013; Dwivedi et al., 2010) recovered from resources such as
250
deep-sea sediments, sedimentary rock samples.
251 252 11
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253 254
3.2. Optimization of parameters
255
3.2.1. Effects of pH
256
Mn bioleaching by Aspergillus oryzae was studied in the pH range 2-8 in 2 % Mn
257
concentration. An increase in biomass growth was observed at pH ~5-6 (Fig.1) and the fungal
258
strain showed highest percentage of Mn solubilization at pH ~6.The growth of mycelium is
259
observed with a varied range of pH 5-6 (Fig. 1). It is clear that bioleaching is directly related to
260
the biomass growth. At optimum pH, growth is maximum so metabolic activity is maximum
261
which leads to better Mn leaching. (Acharya et al., 2003) reported elevation in Mn removal by
262
Aspergillus niger with decrease of pH. (Gholami et al., 2010) suggested high metal binding
263
activity with increase in pH due to decrease in proton concentration, as they also compete for
264
the binding sites. Most of the reducing microorganisms grow optimally at pH 5 to 6.5 because
265
primary acidic situations produce tough reducing atmosphere and the microbes can uphold
266
homeostasis at pH 6.5 avoiding intracellular damage (Acharya et al., 2003).
267
The highest observable growth in addition to Mn reduction was calculated at pH 3 with
268
highest efficiency in 4th day of leaching. However, various forms of Mn are soluble with a
269
varied range of pH and are observed to be usually mobile in water and soil environs (Li et al.,
270
2013). According to (Roane and Pepper, 1999), pH of the system may have considerable
271
solubilizing effect on heavy metal and henceforward their metal bioavailability. As reported by
272
(Mehta et al., 2010) the effective impact of microbial inhabitants by distressing their
273
development, external feature, bio-chemical processes eventually observed subsequent decrease
274
in their pH and other extracellular activities (Roane and Pepper, 1999).
275
12
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276 277
3.3.2. Effect of pulp density
278
The effect of initial concentration on Mn solubilization by Aspergillus oryzae was examined
279
using pulp density ranging from 2 % to 6 %. Substantial Mn solubilization occurred over the
280
entire Mn concentration range studied (Fig. 2). Maximum biomass growth of fungal mycelium
281
was observed at lower Mn initial concentrations of 2 % after 20 days. At pulp density 2 % and
282
6%, 2.119 gm/L and 0.533 gm/L of fungal biomass growth was recorded within 20 days of
283
incubation, respectively showing its relevant growth rate. The resultant toxicity of Mn acquired
284
due to the mutagenic effect on various microbes have been earlier described with concentrations
285
at 5- 10 mg/L of Mn compounds. The above concentration shows an inhibitory effect to almost
286
all fungal mycelium in cultured medium (Amin et al., 2014).
287 288
3.2.3. Effect of carbon sources
289
Microbial solubilization by Aspergillus oryzae of Mn has a significant correlation to carbon
290
dosage. Effect of several carbon sources like sucrose, fructose, dextrose and glucose in Mn
291
bioleaching was investigated. Maximum bleaching was observed in case of dextrose and hence
292
it was chosen for carrying out further experiments (Fig. 3). Maximum growth of fungal biomass
293
weight was obtained with dextrose after 7 days, serving as the effective carbon source for Mn
294
solubilization. It is well known that toxic Mn(II) was transformed to non-toxic Mn(VI) by a
295
microbial mechanism where the electrons are transferred to Mn(II) during bioleaching (Liu et
296
al., 2008). Hence, we can conclude that the carbon source (dextrose) act as electron donor in
297
this method and having the maximum capability of reducing activity of Mn.Thus, microbes
13
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298
result with a highest growth with the respective carbon dosage by addition of different carbon
299
sources (Li et al., 2008).
300
4. Bioleaching study
301
Mn bioleaching capability by fungal mycelium is significantly raised as the strain adapts itself
302
to tolerate high concentration of Mn (Das et al., 2011). The experimental results for bioleaching
303
showed 79 % of Mn recovery by Aspergillus oryzae (KP309810) within 20 days of leaching
304
study. The process of bio-oxidation and bio-reduction by bioleaching of Mn by various fungi
305
involves a significant method for the development of insoluble Mn (III, IV) in the expected
306
surroundings as depicted by (Gadd, 2010). Metal bioleaching by heterotrophic fungal strains
307
usually comprise an indirect method with macrobiotic generation of natural acids (gluconic
308
acid, lactic acid, oxalic acid, citric acid), some assimilated amino acids, and other simpler
309
metabolites (Das et al., 2012). As projected by (Behera et al., 2015), the intercellular
310
mechanism of Aspergillus sp. involves the conversion of sugar compounds (sucrose, glucose)
311
into different organic acids leading in decreasing the pH of the medium. The generated
312
metabolic products generally have the diplomatic consequence of dissolving insoluble metal
313
complexes from minerals by reducing the level of pH and rising concentration of soluble metals
314
by complexing (Bahh et al., 2013).
315
However various group of fungi are being involved in boosting oxidation of Mn which is
316
probably due to enzymatic and non-enzymatic interaction with the fungal metabolic product.
317
(Chen et al., 2014) accounted that Mn ion is measured as the most significant metal for cellular
318
metabolic activity. Thus, the existence of Mn ions accelerates the activation of enzymatic action
319
over elevation of oxalate compound. The cofactor of Mn is positioned in the cytoplasmic area of
14
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320
Asperigillus niger, afterward it metabolizes the alteration of oxalo-acetate to acetate and oxalate
321
(Han et al., 2013).
322 323
5. Microscopic analysis
324
The SEM micrographs of Mn ore surface leached and unleached with fungal mycelium cells
325
are depicted in Fig. 4. Morphology observed after SEM analysis reveals that the surface of Mn
326
ore is smoother than that of ore surface exposed to fungal leaching. However, SEM image of
327
1000 X ore particles vary largely in arrangement of crystals viewed in the given figure. In the
328
other hand, a big sum of deposits in a pattern of comparative free porous, rough, coagulated
329
precipitates are observed on the revealed surface of leached sample ore treated with fungus after
330
20 days of bioleaching (Das et al., 2015a). It is of course demonstrated that the ore treated with
331
fungus views a fraction of bulky quartz particles. Chemical and metallic constituents were
332
determined by using an EDX spectrometer in (Fig. 4 C, D, E). Micrographs illustrate the shape
333
of the ore particles and results suggest that the ore was a heterogeneous material containing a
334
large particle size distribution (Fig. 4 A, B). Analysis of the particles with EDX indicates that
335
Mn along iron, aluminum and silicon are present in the ore sample. Similar studies on
336
morphological characters and elemental chemical analysis of the ore from chromite mines (Das
337
and Mishra, 2010; Xin et al., 2010; Zhang et al., 2008).
338 339
6. Estimation of total protein and protein profiling
340
The total protein content of fungal strains Aspergillus oryzae was found to be 28.38 g/l. The
341
visible molecular mass of fungal strains after SDS-PAGE analysis produced a multiple band of
342
protein. It is evident from the figure that the bands obtained with separated protein according to
15
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343
their molecular weight as 116, 97, 66, 55, 36 and 14.2 kilo Dalton (kDa) protein bands were
344
observed with more thickness in presence of Mn. Aspergillus oryzae contains 20 protein bands
345
with the minimum band from 14.2 to 97 KDa (Fig. 5). The alteration of protein bands of fungal
346
species after treatment with Mn ore may be obtained due to bioleaching activity beyond the
347
optimum temperatures or attributed due to denaturation of the proteins involved in the oxidizing
348
system of the fungus caused by the increase in the rate of thermal death of the microorganisms
349
(Durve et al., 2013). The changes in the conformation of protein patterns is basically because
350
Mn ion may alter the biological membranes, extracellular and intracellular enzyme specified
351
mutation and damage the cellular and structural variabilities (Roane and Pepper, 1999).
352
In observing the effect of the toxicity of metal ions onto adequate exposure of metal, the
353
sensitivity of microbe-metal interaction will outcome with instant death of the cellular
354
components. Due to their damage in the essential components of cell, inclined changes in the
355
outline of population dimensions are acquired (Wyatt, 2015).
356 357
7. Fourier transform infrared spectroscopy (FTIR) analysis
358
FTIR analysis was carried out using model FTIR-4100, JASCO instrument. The IR spectrum of
359
Aspergillus oryzae mycelium cells without Mn and IR spectrum of all the three fungus with Mn
360
ore treated was documented in the spectrum ranging (4000 and 400 cm-1) (Table 1).
361 362
7.1. Characterization of MnOx
363
Collected low grade Mn ore samples were grounded and powdered finely and analyzed for
364
FTIR analysis and their peaks were obtained at 1099.80 cm-1, 582.40 cm-1, 476.33 cm-1, 417.51 cm-1
16
ACCEPTED MANUSCRIPT
cm-1
and 476.33
cm-1
365
(Fig. 6 A). The acquired peaks observed at 582.40
366
stretching and CH2 vibrations as described by (Buciuman et al., 2005).
is recognized as MnOx
367 368
7.2. FTIR Spectral characterization of fungal cells (Mn+/Mn-)
369
Infra-red (IR) spectrum of dry biomass cells of Aspergillus oryzae (Fig. 6 B) and Mn ore treated
370
Aspergillus oryzae were obtained in (Fig. 6 C). The observed major variation of peaks obtained
371
by untreated at 1631.48 cm-1, 1089.58 cm-1, 775.24 cm-1, 688.46 cm-1and 464.76 cm-1 (Table 2). The
372
above obtained peaks generally understand the probable metal-cell interaction that codes for
373
functional groups like C=C group (Amide I), C-N stretching (Aliphatic amines), N-H wag
374
(primary (10) and secondary (20) amines), -C=C- stretching (alkynes) and CH2 vibrations
375
(Polysaccharides) respectively (Table 3). While on the other hand the spectrum of Mn treated
376
Aspergillus oryzae shifted the peaks to 3384.46
377
1377.89
378
corresponding to phenols, alcohols compounds, C=C group (alkanes), -C=C stretch (amide I), -
379
C-O stretch (alcohols, carboxylic acid,). The changes in C-N stretching compare with aliphatic
380
amines, while CH2 vibration correlates to polysaccharides (Parikh and Chorover, 2005).
cm-1,
1249.65
cm-1
and 569.86
cm-1.
cm-1,
2926.45
cm-1
1650.77
cm-1,
1545.67
cm-1,
These peaks represent -OH group C-H stretching
381
Formation of newer carbon (C) and nitrogen (N) bonds in Mn treated fungal species is
382
believed to change the conformation of protein patterns. The change in corresponding peaks
383
suggests conformational alteration in protein amide patterns due to fungal bioleaching that may
384
be acquired because of enzymatic activity (Roy et al., 1994). Biomining remediation of Mn (II),
385
utilizing fungal mycelium has been observed in biologically intracellular metal accumulation
386
converting the functional groups using direct bio-transformation with functional metabolites.
387
(Kalinowska et al., 2014) carried out the FTIR spectroscopic analysis providing instantaneous
17
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388
molecular level metabolism through their organic and inorganic dynamic interaction with the
389
metal surface. FTIR also reveals the study of various groups associated with lipid,
390
carbohydrates and proteins during microbe-mineral alteration evaluated with physiological
391
changes.
392
8. Possible mechanism for variation in protein expression
393
In most instances, some fungal species have also been reported to promote Mn oxidation due to
394
interaction with metabolites i.e bio-generated acid generated from fungal cell component. The
395
capability of indigenous microbes to tolerate Mn heavy metals is sustained by different basic
396
mechanisms which comprise of intracellular enzymatic alteration, bioreduction in sensitivity of
397
cellular surfaces, metal and cellular elongation. On the other hand, oxidation resulted may be
398
due to extracellular presence of Mn(II) peroxidase. Other additional enzymes for altering the
399
expression of protein patterns such as cellobiose dehydrogenase (CDH), may predominantly
400
participate in reduction and oxidation cycling of Mn (Nealson et al., 2006) confirmed that the
401
enzyme cellobiose dehydrogenase can decrease the concentration of insoluble MnO2 and
402
generate Mn(II), the substrate for MnP. Several investigators (Nealson, 2006; Vrind et al., 1986)
403
have examined that Mn(II) oxidizing microorganisms can transfer Mn oxides as an e- acceptor
404
for the continued existence under anaerobic or microaerophili conditions. The enzyme
405
manganese peroxidases secreted by fungus are utilized to catalyze the bio-transformation of
406
Mn(III) compounds that can also consequently oxidize the bioleaching activity and altering the
407
protein patterns ultimately (Das et al., 2015). We can thus conform that different fungal strains
408
participates in enhanced mechanism of Mn biomass interaction will come forward as important
409
mineral large scale remediation due to their higher tolerance of acute conditions of
410
contaminated mining sites.
18
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411 412
9. Conclusions
413
Bioleaching is an economic process of recovering minerals from low to medium grade ores and
414
waste residues using microorganisms. As there is no substitute for Mn in its major application,
415
the demand for Mn is ever increasing due to rapid industrialization and gradual depletion of
416
high grade ore and the rising insufficiency of natural deposits. The conventional pyro-
417
metallurgical methods used for extraction of Mn from are very expensive, requires lots of
418
energy and additionally causes environmental pollution problems Bioleaching is considered as a
419
persuading way to solve these problems. In the current investigation leaching of Mn from
420
mining waste ore using native isolated fungi Aspergillus oryzae was investigated. Influence of
421
operational conditions on bioleaching of Mn ore with Aspergillus sp. were examined for
422
duration of 20 days. The optimal conditions for enhanced recovery of Mn (79 %) were observed
423
to be at temperature 37 0C, pH 6 and pulp density 2 % (w/v). Increasing metal concentrations
424
seem to be the reason behind the shifting in proteomic data of the fungal cells during the
425
bioleaching procedure. Mn and protein interaction results in the alteration of amide
426
configuration and may also modify the conformational changes in functional groups due to Mn
427
solubilization.
428
It is very clear that proteins play a significant role in mineral-microbe interphase with ores.
429
The present study of deviation of protein expression and conformation had been valuable in
430
supervising the modification that occurs in the fungal cells during bioleaching. Study of
431
advance contact mechanisms are needed to recognize specific proteins concerned in the
432
bioleaching procedure so that the information can pave a way for improved biorecovery of
433
metals from their ores.
19
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434 435
Acknowledgment
436
Authors express their thanks to the (DBT) Department of Biotechnology Technology,
437
Government of India (BT/PR7454/BCE/8/949/2012), Department of Science and Technology
438
(DST) (SP/YO/031/2016) and Ministry of Mines Government of India (07/46SSAG/CAT) for
439
granting projection in manganese biomining and recycling studies.
440
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441
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Bioleaching of uranium by aspergillus niger and aspergillus terreus isolated from uraniferous
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5. Banh, A., Chavez, V., Doi, J., Nguyen, A., Hernandez, S., Ha, V., Jimenez, P., Espinoza, F.
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Fig. 1. Effect of pH on Mn solubilization by A. oryzae
1
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Fig. 2. Effect of pulp density on Mn solubilization by A. oryzae
2
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Fig. 3. Effect of carbon sources on Mn solubilization by A. oryzae.
3
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Fig. 4. Microscopic analysis of treated and untreated ore samples through SEM/EDX (A) SEM analysis of Mn ore samples (B) SEM analysis of fungal treated ore sample with magnification of 1000 X (C) photomicrograph of EDX with electron image at 100um (D) EDX images of Mn ore sample with elevated peaks of elements like
4
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Fig. 5. Conformational protein patterns by SDS PAGE of Mn ore treated and untreated Asperigillus orzyae (MSF 7)
5
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Fig. 6. Fourier transform infrared spectroscopy (FTIR) peaks in cm-1 (A) IR spectra of low grade Mn ore (B) IR peaks of Aspergillus oryzae (C) IR peaks of Mn ore treated Aspergillus oryzae.
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Fig. 5. Conformational protein patterns by SDS PAGE of Mn ore treated and untreated Asperigillus orzyae (MSF 7)
Table 1: FT-IR analyzed wave number frequencies in cm-1 and their assigned groups
Frequency (cm-1)
ACCEPTED MANUSCRIPT Functional group Assigned
Below 500 cm -1
C-C bending vibrations occur at very low frequencies.
750-200 cm -1
MnOx stretching, bending, and wagging vibrations
650-450 cm -1
CH2 vibrations of polysaccharides
1200- 800 cm -1
C-C stretching vibrations are weak and in this region.
1439-1399 cm -1
C-H bending vibrations methyl groups.
1340- 1400 cm -1
Overlapping doublets for t-butyl and isopropyl group
3000-2840 cm -1
C-H stretching in the alkanes.
2962 & 2872 cm -1
Saturated hydrocarbons containing methyl groups.
1
Table 2 IR spectra peaks of Mn ore sample
Peaks values
Bond assigned MANUSCRIPT Assignment ACCEPTED
Functional group
(cm-1) 1099.80
C-N stretching
(C-N)ar
Aliphatic amines
582.40
CH2 vibrations
(CH2)ar
Polysaccharides
476.33
MnOx stretching, bending, and
(MnOx)
a-Mn2O3
(MnOx)
b-MnO2
wagging vibrations 417.51
MnOx stretching, bending, and wagging vibrations
Table 3. IR peaks in cm-1of Aspergillusoryzaeand Mn ore treated Aspergillusoryzae Aspergillusoryzae Peaks values (cm-1)
Bond
Assignment
1631.48
C=C group
v(C═O)
1089.58
C-N stretching N-H wag
(C-N)ar
775.24
688.46 540.94 464.76
Mn ore treated Aspergillusoryzae Functional group
Peaks values (cm-1)
Bond
Amide I: 3384.46 -OH group ACCEPTED MANUSCRIPT C═O, C═ N, N═ H Aliphatic amines 2926.45 C-H stretching
Assignment
Functional groups
v(OH)ar
Phenols, alcohols
(CH)ar
Alkanes
(N-H)ar
Primary (1O) Secondary(2O) amines
1650.77
C=C group
v(C═O)
Amide I: C═O, C═ N, N═ H
-C=Cstretch CH2 vibrations
v(C═C)
alkynes
1545.67
-C=C stretch
v(C═C)-c=c–
(CH2)ar
Polysaccharides
1377.89
-C-O stretch
v(COO−)
CH2 vibrations
(CH2)ar
Polysaccharides
1249.65
C-N stretching
(C-N)ar
Amide I: N═H, C═ N Alcohols, Carboxylic acid, esters, ethers Aliphatic amines
569.86
CH2 vibrations
(CH2)ar
Polysaccharides
a,c