Treatment of alkaline Cr(VI)

27 Pages·2015·2.8 MB·English

AEM Accepted Manuscript Posted Online 5 June 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.00853-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Treatment of alkaline Cr(VI) contaminated leachate with an alkaliphilic metal-reducing bacterium
2 Mathew P Watts1a, Tatiana V. Khijniak2, Christopher Boothman1 & Jonathan R. Lloyd1*
3 1School of Earth, Atmospheric and Environmental Sciences and Williamson Research Centre for
4 Molecular Environmental Science, University of Manchester, UK; 2Institute of Microbiology, Russian
5 Academy of Sciences, 7/2 Prospect 60-letiya Oktyabrya, Moscow 117312, Russia D
o
w
6 aCurrent address: School of Earth Sciences, University of Melbourne, Victoria, 3010, Australia. n
lo
a
d
7 *[email protected] e
d

f
r
8 o
m

9 Abstract h
t
t
p
10 Chromium in its toxic Cr(VI) valence state is a common contaminant particularly associated with :/
/
a
e
11 alkaline environments. A well publicized case of this occurs within Glasgow, UK, where poorly m
.
a
12 controlled disposal of a cementitious industrial by-product, chromite ore processing residue (COPR), s
m
13 has resulted in extensive contamination by Cr(VI) contaminated alkaline leachates. In the search for .o
r
g
14 viable bioremediation treatments for Cr(VI), a variety of bacteria have been identified that are /
o
n
15 capable of reduction of the toxic and highly soluble Cr(VI) to the relatively non-toxic and less mobile J
a
n
16 Cr(III) oxidation state, predominantly under circum-neutral pH conditions. Recently, however, u
a
r
17 alkaliphilic bacteria have been identified that have the potential to reduce Cr(VI) under alkaline y

1
0
18 conditions. This study focuses on the application of a metal-reducing bacterium to the remediation of ,

2
0
19 alkaline Cr(VI) contaminated leachates from COPR. This bacterium, belonging to the Halomonas 1
9

b
20 genus, was found to exhibit growth concomitant to Cr(VI) reduction under alkaline conditions (pH
y

g
21 10). Bacterial cells were able to rapidly remove high concentrations of aqueous Cr(VI) (2.5 mM) u
e
s
22 under anaerobic conditions, up to a starting pH of 11. Cr(VI) reduction rates were controlled by pH, t
23 with slower removal observed at pH 11, compared to pH 10, while no removal was observed at pH
24 12. The reduction of aqueous Cr(VI) resulted in the precipitation of Cr(III) biominerals, which were
25 characterized using TEM-EDX and XPS. The effectiveness of this haloalkaliphilic bacteria for Cr(VI)
26 reduction at high pH, suggests potential for its use as an in situ treatment of COPR and other
27 alkaline Cr(VI) contaminated environments.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/

o
n

J
a
n
u
a
r
y

1
0
,

2
0
1
9

b
y

g
u
e
s
t
28 Introduction
29 Chromium (Cr) is a significant component of contaminated soil and groundwater, through a variety of
30 environmental exposures from its widespread use in metallurgy and industrial processes (1-3).
31 Under most environmental conditions it is stable as the Cr(VI) and Cr(III) valence states (4). The
32 Cr(III) state dominates under reducing conditions, forming largely insoluble Cr(III) hydroxide phases D
o
33 (5, 6), which are widely considered non-toxic (7). In contrast, the Cr(VI) species dominates under w
n
34 oxidizing conditions, forming the toxic, carcinogenic and highly soluble oxyanions; HCrO4-, CrO42- loa
d
35 and Cr2O42- (8, 9). Due to the greater stability and mobility of Cr(VI) at high pH, it is particularly ed

f
r
36 associated with contaminated alkaline environments (10). A well known example of alkaline Cr(VI) o
m

37 contamination relates to the poorly controlled disposal of waste from the “high lime” chromite ore h
t
t
p
38 (FeCr2O4) processing technique; chromite ore processing residue (COPR) (11, 12). Roasting of the ://
a
e
39 chromite ore with lime causes oxidation of Cr(III) to Cr(VI), enabling leaching with water (12). m
.
a
40 However due to inefficiencies in the process, COPR contains significant concentrations of Cr, s
m
41 typically 3-7% by mass, of which 1-30% is typically in the Cr(VI) state (13, 14), while the addition of .o
r
g
42 lime produces typically high pH values of 11-13 (15). The Cr(VI) forms part of a complex mineralogy, /
o
n
43 which, upon saturation with groundwater readily yields alkaline leachate with high concentrations of J
a
n
44 aqueous Cr(VI) (15-17). COPR related contamination is a global issue, with significant cases u
a
r
45 reported in the UK, USA, Eastern Europe, India, Pakistan and China (11, 12, 18). For example, in y

1
0
46 Glasgow, UK, the poorly controlled disposal of >2 million tonnes of COPR has resulted in ,

2
0
47 widespread contamination with highly alkaline Cr(VI) leachate, with ground and surface waters 1
9

48 containing up to 100 mg L-1 (16, 19-21). These values are far in excess of the World Health b
y

g
49 Organizations upper limit for Cr(VI) in drinking water; 0.05 mg L-1 (22). u
e
s
t
50 A potential treatment of Cr(VI) contamination involves harnessing the microbial metabolism of
51 bacteria which are capable of enzymatic metal reduction, reducing Cr(VI) to the relatively insoluble
52 Cr(III) (23, 24). The ability to enzymatically reduce Cr(VI) has been observed among a diverse range
53 of bacteria (25, 26), primarily among the facultative anaerobes (27). Microbial Cr(VI) reduction is
54 often attributed to enzymes which serve alternative metabolic functions (28), while a restricted range
55 of bacteria are capable of using Cr(VI) as the terminal electron acceptor for growth (29, 30). Most
56 previous studies have been carried out at near-neutral conditions and extremes of pH have proved a
57 major limiting factor to enzymatic reduction (31). As Cr(VI) contamination is primarily associated with
58 alkaline environments (4), several studies have sought to culture alkaliphilic bacteria capable of
D
o
59 Cr(VI) reduction at high pH (32-37). Alkaliphiles exhibit optimum growth under alkaline conditions w
n
60 (pH 9 – 12) (38), while a number of these, the haloalkaliphiles, also require salinity for optimum lo
a
d
61 growth (39). Haloalkaliphiles of the Halomonas genus are especially well represented in high pH and e
d

62 high salt environments (40, 41), and a number of studies have found these organisms to be capable fr
o
m
63 of Cr(VI) reduction (34, 36). Halomonas species have also been reported for other remedial
h
t
t
64 reactions, such as the reduction of nitrate (42), while the isolate used in this current study was also p
:
/
/
a
65 able to reduce the nuclear contaminant Tc(VII) to Tc(IV) (43). e
m
.
a
66 Despite the identification of a small number of alkaliphilic bacteria capable of reducing Cr(VI) in s
m
67 model laboratory solutions, there remains a need to test these bacterial systems against .o
r
g
68 environmental Cr(VI) contamination. The aim of this study is to determine if the microbial metabolism /
o
n
69 of alkaliphilic bacteria can be harnessed for the reductive precipitation of Cr(VI) in high pH leachates J
a
n
70 of COPR, modified with haloalkaliphilic medium and bacteria.. This was explored by using a u
a
r
71 haloalkaliphilic soda lake isolate which has previously been reported to be effective at high pH y

1
0
72 reduction of Tc(VII) (43). The findings of this study would therefore represent the first investigations ,

2
0
73 in to the direct treatment of COPR leachates using an alkaliphilic Cr(VI) reducing bacterium. 1
9

b
74 y

g
u
e
75 Experimental section s
t
76 Organism and culture conditions. A culture of the facultative anaerobic haloalkaliphilic bacterium,
77 isolated from Mono Lake (California, USA), herein referred to as the Mono isolate, by N.N. Lyalikova
78 (Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia). The isolate was cultured
79 using sterile anaerobic growth media (pH 10) consisting of a basal media of 13 g L-1 Na CO , 4 g L-1
2 3
80 NaHCO , 50 g L-1 NaCl and 0.5 g L-1 K HPO , and the following additional growth nutrients; 0.1 g L-1
3 2 4
81 MgSO •7H O, 0.1 g L-1 NH Cl, 2 g L-1 Na-acetate, 2 g L-1 yeast extract and 2 mL of Mineral Elixir
4 2 4
82 (2.14 g L-1 nitrilotriacetic acid, 0.1 g L-1 MnCl •4H O, 0.3 g L-1 FeSO •7H O, 0.17 g L-1 CoCl •6H O,
2 2 4 2 2 2
83 0.2 g L-1 ZnSO •7H O, 0.03 g L-1 CuCl •H O, 0.005 g L-1 AlKSO •12H O, 0.005 g L-1 H BO ,0.09 g
4 2 2 2 4 2 3 3
84 L-1 Na MoO , 0.11 g L-1 NiSO •6H O and 0.02 g L-1 Na WO •2H O).
2 4 4 2 2 4 2
D
o
85 COPR sample collection and preparation of a COPR extracted medium. A sample of COPR was w
n
lo
86 obtained from a borehole at a site in the south east of Glasgow, UK, transferred to a sterile plastic a
d
e
87 container and stored in the dark for approximately 2 years at 10⁰C until use. The COPR has been d

f
r
88 extensively characterized in a previous study and found to be composed of a cementitous o
m

89 mineralogy, with considerable leachable Cr(VI) content (44, 45), consistent with previous studies (15, h
t
t
p
90 17). To prepare the COPR extract medium, a sub-sample of field moist COPR was added to the :/
/
a
e
91 basal medium at 5:100 (w/v) for growing cell and 10:100 (w/v) for resting cell experiments, under m
.
a
92 aerobic conditions in the absence of any growth nutrients. The COPR medium slurry was s
m
.
93 homogenized by shaking and left in the dark at 20⁰C for 24 hours to equilibrate. The resulting slurry o
r
g
/
94 was then filter sterilized using a 0.22 µm filter. o
n

J
95 Growing cell experiments. Experiments using a growing culture of the Mono Lake isolate were a
n
u
96 conducted by inoculation of the previously stated 5:100 (w/v) COPR : basal medium extract, in the a
r
y

97 presence of added growth nutrients. Growing cell cultures were prepared by mixing 11 mL of the 1
0
,

98 filter sterilized 5:100 COPR extract with 3.5 mL of sterile growth medium in sterile 20 mL serums 2
0
1
99 bottles, which were then sealed using butyl rubber stoppers and aluminum crimps, in equilibrium with 9

b
y
100 air. The serum bottles were then inoculated with a 1 mL aliquot of a growing anaerobic culture,
g
u
101 maintained at 20⁰C, of the Mono Lake isolate. The incubations were then incubated at 30⁰C in the e
s
t
102 dark for the duration of the experiment. Samples (1 mL) were removed using a N degassed syringe
2
103 and centrifuged (Sigma 1-14 Microfuge) at 13000 g for 5 minutes, the supernatant was then
104 removed for Cr(VI) analysis. The remaining solids were re-suspended in 1% NaCl and again
105 centrifuged (Sigma 1-14 Microfuge) at 13000 g for 5 minutes, the supernatant discarded and
106 replaced with 100 µL of 1% NaCl and homogenized for protein analysis.
107 Resting cell experiments. The isolate was also used in a series of anaerobic resting cell
108 experiments in the presence of the 10:100 (w/v) COPR : basal medium extract, as detailed in the
109 medium preparation section. Under aerobic conditions, growth medium was inoculated with a culture D
o
110 of the Mono Lake bacterium and incubated in a sterile Erlenmeyer flask on a shaking incubator at 30 w
n
lo
111 ⁰C. The culture was then harvested, in late log phase (approximately 24 hours), using a centrifuge a
d
e
112 (Sigma 6k15) at 5000 g for 20 minutes and washed 3 times using basal medium under an N2 d

f
r
113 atmosphere. The washed cells were then used to inoculate resting cell experiments to a final protein o
m

114 concentration of 81.5 µg mL-1 (equivalent to an optical density at 600 nm of 0.45). The resting cell h
t
t
p
115 experiments were composed of 20 mL of the COPR extract medium, supplemented with 2 g L-1 Na- :/
/
a
e
116 acetate and 2 g L-1 yeast extract. All cultures were contained in sterile serum bottles sealed using m
.
a
117 butyl rubber stoppers and aluminum crimps and degassed using N2 gas passed through a 0.22 µm s
m
.
118 filter. Resting cell experiments were established at pH 10, 11 and 12, in triplicate, alongside non- o
r
g
119 inoculated abiotic controls. The starting pH of the medium was 12, and it was subsequently adjusted /
o
n

120 to pH 11 and pH 10 in the corresponding cultures, using sterile 3M HCl. Aqueous samples were J
a
n
121 removed to monitor the geochemical parameters of the experiment using an N degassed syringe u
2 a
r
y
122 and centrifuged (Sigma 1-14 Microfuge) at 13000 g for 5 minutes, a sub-sample of the supernatant
1
0
123 was then analyzed for aqueous Cr(VI) concentration. ,
2
0
1
124 Calculation of Cr(VI) removal reaction rates. The Cr(VI) concentration data was subject to fitting 9

b
y
125 with a pseudo-1st order reaction rate model. The reaction rate is proportional to the concentration of
g
u
126 aqueous Cr(VI) while the cell concentration is assumed to remain in far excess and constant e
s
t
127 throughout the experiment:
128
(cid:1856)[Cr(cid:4666)VI(cid:4667)]
=−(cid:1863) [Cr(cid:4666)VI(cid:4667)]
(cid:1856)(cid:1872) (cid:3042)(cid:3029)(cid:3046)
129
130 where [Cr(VI)] is the concentration of aqueous Cr(VI), t is time and k is the observed 1st order
obs
131 reaction rate constant. The 1st order reaction rate model was only applied where significant aqueous
132 Cr(VI) removal (>50%) was observed.
133 Aqueous phase analysis. The pH of the samples was measured using a meter (Denver Instrument D
o
w
134 UB-10) and a probe (P Cole Parmer 5990-45 CCP), calibrated using relevant pH buffers. The n
lo
135 aqueous Cr(VI) concentration was determined by the 1,5-diphenylcarbazide (DPC) UV-vis a
d
e
136 spectrophotometric method and compared to K CrO standards of known Cr(VI) concentration (46). d
2 4
f
r
o
m
137 Protein assay. Protein concentrations were determined using a bicinchoninic acid (BCA) and

h
t
138 Cu(II)SO4 spectrophotometric assay (47), quantified by comparison to bovine serum albumin (BSA) tp
:
/
/
139 standards. All UV-vis measurements were recorded on a Jenway 6715 UV/Vis Spectrophotometer. a
e
m
140 Solid phase analysis. At the end of the resting cell experiment, the replicates which exhibited Cr(VI) .a
s
m
141 removal, were sampled for solid phase analysis. The aqueous slurry was centrifuged (Sigma 1-14 .
o
r
g
142 Microfuge) at 13000 g for 5 minutes, the supernatant removed and replaced with 18.2 MΩ water and
/

o
n
143 homogenized. This was repeated 3 times and the resulting pellet dried in an anoxic glove box prior

J
a
144 to analysis. n
u
a
r
145 For transmission electron microscopy (TEM) imaging, the pellet was re-suspended in ethanol and y

1
0
146 droplets placed upon an Agar Scientific Holey Carbon Film grid and allowed to dry. The TEM ,

2
0
147 analysis was performed on a Philips CM200 FEG TEM equipped with a field emission gun (FEG) 1
9

148 and energy dispersive X-ray analyzer (EDX), Oxford Instruments X-max 80 mm2 SDD INCA EDX. b
y

g
u
149 X-ray photoelectron spectroscopy (XPS) was performed on a Kratos Axis Ultra spectrophotometer e
s
t
150 with a monochromated Al Kα X-ray source. Analysis was carried out with an analyzer pass energy of
151 80 eV (wide scans) and 20 eV (narrow scans) with a total energy resolution of 1.2 and 0.6 eV
152 respectively, at a base pressure of 5 × 10−10 mbar. All spectra were fit with a Shirley background
153 model (48) and had their photoelectron binding energies (BE) referenced to the C 1s adventitious
154 carbon peak (285 eV BE). Fitting of the Cr 2p region was conducted using 70 % Lorentzian and 30
155 % Gaussian curves.
156 DNA extraction, PCR amplification and 16S rRNA gene sequencing. The DNA of the isolate was
157 extracted using a PowerSoil DNA Isolation Kit (PowerSoil DNA Isolation Kit, MO BIO Laboratories
158 INC, Carlsbad, CA, USA). The DNA was amplified by polymerase chain reaction (PCR) using D
o
159 several broad specificity 16S rRNA gene primers, to obtain overlapping amplified 16S rRNA w
n
lo
160 fragments, including; 8F (49), 530F (50), 519R (51), 943R (50) and 1492R (51). a
d
e
161 The dideoxynucleotide method was used to determine the nucleotide sequences (52), using an ABI d

f
r
o
162 Prism BigDye Terminator Cycle Sequencing Kit in combination with an ABI Prism 877 Integrated m

h
163 Thermal Cycler and ABI Prism 377 DNA Sequencer (Perkin Elmer Applied Biosystems, Warrington, t
t
p
:
164 UK). A contig was generated from the sequences (typically 900 base pairs in length), using DNA //
a
e
165 Dragon v1.6 (SequentiX Digital DNA Processing, Klein Raden, Germany). The consensus sequence m
.
a
s
166 (1446 base pairs in length) was analyzed against the NCBI (USA) database using BLAST program m
.
o
167 packages and matched to known 16S rRNA gene sequences. r
g
/

o
168 The phylogenetic tree was constructed using MEGA5 (53). The evolutionary history was inferred n

J
169 using the Neighbor-Joining method (54) and the evolutionary distances were computed using the a
n
u
170 Maximum Composite Likelihood method (55). a
r
y

1
171 0
,

2
0
172 Results and discussion 1
9

b
173 Phylogenetic characterization. Sequencing of the 16S rRNA gene of the Mono Lake isolate y

g
u
174 showed that the organism belongs to the Halomonas genus of the γ-Proteobacteria (Fig. 1). The e
s
t
175 isolate occupies a clade with Halomonas mongoliensis, and shares 16S rRNA gene sequence
176 similarity to a variety of Halomonas species, see Fig. 1.
177 Halomonas species are often highly represented in isolates from hypersaline and alkaline
178 environments such as soda lakes (40, 41). These obligate heterotrophs have gained attention for
179 other potentially useful behavior, such as their ability to degrade aromatic compounds (56-58) and
180 produce alkaline enzymes with possible biotechnological applications (59, 60). In addition to this, a
181 number of Halomonas species, including H. mongoliensis (61), the closest phylogenetic match to the
182 Mono Lake;, have been found to be capable of anaerobic nitrate reduction under alkaline (pH 10)
183 and high salt conditions (4M Na+) (36). Several closely related Halomonas strains have also been
D
o
184 found to be capable of alkaline Cr(VI) reduction under anaerobic conditions (34, 36). As the Mono w
n
185 Lake isolate studied here has also been shown to reduce Tc(VII) to Tc(IV) (43), it may potentially lo
a
d
186 reduce a variety of redox active metals. e
d

f
r
187 Cr(VI) reduction during growth. Upon inoculation of aerobic COPR extract growth medium, with o
m

188 the Mono Lake Halomonas species, the aqueous Cr(VI) concentration decreased to below detection h
t
t
p
189 limits within 170 hours (Fig. 2), while the pH was maintained at 10 throughout the experiment. :/
/
a
e
190 Concurrent to this removal of Cr(VI), protein concentrations increased steadily over the reaction m
.
191 period reaching over 100 µg mL-1 after 168 hours of incubation. This concurrent increase in protein as
m
192 levels, while Cr(VI) decreased, is clear evidence for growth of the isolate in the presence of .o
r
g
193 significant Cr(VI) concentrations. As these cultures were initially incubated under oxic conditions, /
o
n
194 where the facultative anaerobic Halomonas species likely consumes O2 as an initial electron J
a
n
195 acceptor, it is unclear if growth is directly coupled to Cr(VI) reduction u
a
r
y
196 Cr(VI) reduction by resting cells. To identify the impact of pH on the kinetics of Cr(VI) reduction, a 1
0
,
197 series of resting cell experiments were conducted at pH 10, 11 and 12 (Fig. 3). The Cr(VI) 2
0
1
198 concentrations of cell-free abiotic control experiments showed little change over the time of the 9

b
199 experiment at all pH values, remaining at ~2500 µM throughout. However, rapid Cr(VI) removal was y

g
u
200 noted in the presence of cells of the Halomonas sp. in the cultures poised at pH 10 and 11, along e
s
t
201 with reductive precipitation of Cr(III), confirmed by XPS analysis of the resulting precipitates (Fig. 4
202 and Table 1). Thus Cr(VI) removal was due to direct, anaerobic, enzymatic Cr(VI) reduction by the
203 Mono Lake Halomonas isolate.
204 The solution pH was found to have a strong control over enzymatic Cr(VI) reduction, with no
205 appreciable Cr(VI) removal observed in the pH 12 replicate, suggesting a loss of metabolic activity
206 under extremely alkaline conditions. The rapid removal of Cr(VI) noted at pH 10 and 11 translated to
207 k values that were considerably higher at pH 10 (0.0409 hr-1, R2 = 0.96) compared to the pH 11
obs
208 replicate (0.0126 hr-1, R2 = 0.98), see Fig 3c. As the starting pH of 11 evidently buffers down to 10.5
D
o
209 during incubation by addition of the bacterial isolate, it is this level that should be assumed to be the w
n
210 upper limit of sustained Cr(VI) removal observed in this study. Reduction of Cr(VI) at this highly lo
a
d
211 alkaline pH is in line with the upper limits of enzymatic Cr(VI) reduction reported previously (34, 36, e
d

212 62, 63). These values are also consistent with previously reported optimum growth conditions, of pH fr
o
m
213 9-10, for closely related Halomonas species (42, 61). The pH values of the COPR leachate and
h
t
t
214 contaminated groundwater are typically within the range of 9 - 12.5 (16, 64). Therefore, the observed p
:
/
/
a
215 removal of Cr(VI) at alkaline values up to 10.5 indicates that a bioremediation approach using the e
m
.
216 Halomonas species may represent a possible treatment of a proportion of COPR leachates, without a
s
m
217 the need for pH amendment prior to inoculation. While treatment of higher pH COPR leachates .
o
r
g
218 would require some degree of buffering to a lower pH, albeit to a lesser extent than required for
/

o
n
219 bioremediation using neutrophilic bacteria.

J
a
n
220 Characterization of the Cr precipitates. Upon visual inspection of the resting cell incubations at u
a
r
221 the end of the experiments, a purple precipitate was observed in the pH 10 cultures and a green y

1
0
222 precipitate in the pH 11 cultures. The observed color differences in the precipitates would indicate ,

2
0
223 the presence of differing precipitate phases; where Cr(III) minerals can occur as green or purple 1
9

b
224 minerals (65, 66). The fate of the Cr removed from solution in the resting cell experiments was
y

g
225 assessed using TEM-EDX alongside XPS analysis. u
e
s
t
226 The TEM images of the resulting precipitates, and their corresponding EDX spectra, are presented in
227 Fig. 4, for the pH 10 (a to d) and pH 11 (e to h) incubations. The XPS wide scan and Cr 2p region
228 spectra are presented in Fig. 5, along with their elemental composition and Cr valence states
229 presented in Table 1. TEM analysis shows that the precipitates formed at both pH 10 and pH 11