This art icle was downloaded by: [ Nat ional Chung Cheng Universit y] , [ Jyot i Prakash Mait y]
On: 31 August 2011, At : 23: 44
Publisher: Taylor & Francis
I nform a Lt d Regist ered in England and Wales Regist ered Num ber: 1072954 Regist ered office: Mort im er House,
37- 41 Mort im er St reet , London W1T 3JH, UK
Journal of Environmental Science and Health, Part A
Publicat ion det ails, including inst ruct ions f or aut hors and subscript ion inf ormat ion:
ht t p: / / www. t andf online. com/ loi/ lesa20
Biogeochemical characteristics of Kuan-Tzu-Ling,
Chung-Lun and Bao-Lai hot springs in southern Taiwan
Jyot i Prakash Mait y
Kar
b
a b
, Jiin-Shuh Jean
Yen Chen
, Chia-Chuan Liu
b
b
, Bibhash Nat h
, Prosun Bhat t acharya
e
c
, Jochen Bundschuh
, Jiann-Hong Liu
b
b d e
, Shashi B. At la
a
, Sandeep
& Chien-
a
a
Depart ment of Eart h and Environment al Sciences, Nat ional Chung Cheng Universit y, MingShung, Chiayi Count y, Taiwan
b
Depart ment of Eart h Sciences, Nat ional Cheng Kung Universit y, Tainan, Taiwan
c
School of Geosciences, Universit y of Sydney, Sydney, Aust ralia
d
Inst it ut e f or Applied Research, Karlsruhe Universit y of Applied Sciences, Karlsruhe,
Germany
e
KTH-Int ernat ional Groundwat er Arsenic Research Group, Depart ment of Land and Wat er
Resources Engineering, Royal Inst it ut e of Technology (KTH), St ockholm, Sweden
Available online: 31 Aug 2011
To cite this article: Jyot i Prakash Mait y, Chia-Chuan Liu, Bibhash Nat h, Jochen Bundschuh, Sandeep Kar, Jiin-Shuh Jean,
Prosun Bhat t acharya, Jiann-Hong Liu, Shashi B. At la & Chien-Yen Chen (2011): Biogeochemical charact erist ics of Kuan-TzuLing, Chung-Lun and Bao-Lai hot springs in sout hern Taiwan, Journal of Environment al Science and Healt h, Part A, 46: 11,
1207-1217
To link to this article: ht t p: / / dx. doi. org/ 10. 1080/ 10934529. 2011. 598788
PLEASE SCROLL DOWN FOR ARTI CLE
Full t erm s and condit ions of use: ht t p: / / www.t andfonline.com / page/ t erm s- and- condit ions
This art icle m ay be used for research, t eaching and privat e st udy purposes. Any subst ant ial or syst em at ic
reproduct ion, re- dist ribut ion, re- selling, loan, sub- licensing, syst em at ic supply or dist ribut ion in any form t o
anyone is expressly forbidden.
The publisher does not give any warrant y express or im plied or m ake any represent at ion t hat t he cont ent s
will be com plet e or accurat e or up t o dat e. The accuracy of any inst ruct ions, form ulae and drug doses should
be independent ly verified wit h prim ary sources. The publisher shall not be liable for any loss, act ions, claim s,
proceedings, dem and or cost s or dam ages what soever or howsoever caused arising direct ly or indirect ly in
connect ion wit h or arising out of t he use of t his m at erial.
Journal of Environmental Science and Health, Part A (2011) 46, 1207–1217
C Taylor & Francis Group, LLC
Copyright
ISSN: 1093-4529 (Print); 1532-4117 (Online)
DOI: 10.1080/10934529.2011.598788
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
Biogeochemical characteristics of Kuan-Tzu-Ling,
Chung-Lun and Bao-Lai hot springs in southern Taiwan
JYOTI PRAKASH MAITY1,2, CHIA-CHUAN LIU2, BIBHASH NATH3, JOCHEN BUNDSCHUH2,4,5,
SANDEEP KAR2, JIIN-SHUH JEAN2, PROSUN BHATTACHARYA5, JIANN-HONG LIU2,
SHASHI B. ATLA1 and CHIEN-YEN CHEN1
1
Department of Earth and Environmental Sciences, National Chung Cheng University, Ming-Shung, Chiayi County, Taiwan
Department of Earth Sciences, National Cheng Kung University, Tainan, Taiwan
3
School of Geosciences, University of Sydney, Sydney, Australia
4
Institute for Applied Research, Karlsruhe University of Applied Sciences, Karlsruhe, Germany
5
KTH-International Groundwater Arsenic Research Group, Department of Land and Water Resources Engineering, Royal Institute
of Technology (KTH), Stockholm, Sweden
2
Hot springs are the important natural sources of geothermally heated groundwater from the Earth’s crust. Kuan-Tzu-Ling (KTL),
Chung-Lun (CL) and Bao-Lai (BL) are well-known hot springs in southern Taiwan. Fluid and mud (sediments) samples were
collected from the eruption points of three hot springs for detailed biogeochemical characterization. The fluid sample displays
relatively high concentrations of Na+ and Cl− compared with K+, Mg2+, Ca2+, NO2 −, and SO4 2−, suggesting a possible marine
origin. The concentrations of Fe, Cr, Mn, Ni, V and Zn were significantly higher in the mud sediments compared with fluids, whereas
high concentrations of As, Ba, Cu, Se, Sr and Rb were observed in the fluids. This suggests that electronegative elements were
released during sediment-water interactions. High As concentration in the fluids was observed to be associated with low redox (Eh)
conditions. The FTIR spectra of the humic acid fractions of the sediments showed the presence of possible functional groups of
secondary amines, ureas, urethanesm (amide), and silicon. The sulfate-reducing deltaproteobacterium 99% similar to Desulfovibrio
psychrotolerans (GU329907) were rich in the CL hot spring while mesophilic, proteolytic, thiosulfate- and sulfur-reducing bacterium
that 99% similar to Clostridium sulfidigenes (GU329908) were rich in the BL hot spring.
Keywords: Bacteria, geochemistry, hot spring, humic substances, Taiwan.
Introduction
Arsenic (As) and other trace elements including heavy metals are the common chemical constituents of hot spring fluids.[1–4] There are many hot springs distributed worldwide,
e.g., in USA,[2] Turkey,[4] Venezuela,[5] Thailand,[6] Japan,[7]
and Taiwan,[8,9] that are contributing toxic elemental burdens to the earth’s surface and to subsurface groundwater.
In Taiwan, hot springs are classified based on three geological settings, e.g., volcanic hot springs, metamorphic rock
hot springs, and sedimentary rock hot springs.[10] There are
more than 120 hot springs, including thermal springs, cold
Address correspondence to Jyoti Prakash Maity and ChienYen Chen, Department of Earth and Environmental Sciences, National Chung Cheng University, 168 University Road,
Ming-Shung, Chiayi County, 62102, Taiwan; E-mail: jyoti
maity@yahoo.com (J.P. Maity) and yen@eq.ccu.edu.tw (C.Y.
Chen)
springs, mud springs, and seabed hot springs that have been
reported from Taiwan.[11,12]
Volcanic hot springs are generally acidic in nature,
formed during chemical reactions of CO2 and water resulting from volcanic activities. Guishan, Lanyu and Green
Island are acidic volcanic hot springs. Metamorphic rock
hot springs are distributed around the Central and Xueshan
Mountain ranges. Metamorphic rock hot springs generally contain high temperature fluids associated with high
temperature metamorphism. Sedimentary rock hot springs
are distributed on the outer Western regions and Coastal
mountain ranges, including those in Hsinchu, Miaoli, Chiayi, Tainan and Hengchun, as well as Yuli and Taidong
in the eastern coastal mountain ranges. The source of the
sedimentary rock hot springs are the rainwater that seeps
underground and gets heated. The emergence of such water
to the ground surface through a crevasse forms hot springs.
The temperature of these hot springs generally ranges between 30 and 65◦ C.
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
1208
Maity et al.
trace elements in downstream Chianan plain alluvial
aquifers.[20,21] However, limited reports are available on the
biogeochemistry of hot spring fluids in Taiwan.
The aim of this study is to biogeochemically characterize
three hot spring fluids and mud sediments collected from
southern Taiwan. This study further aimed to link geochemical and microbial characteristics of the hot spring
fluids. Microcosm experiments with bacterial isolates from
geothermal fluids were also conducted to investigate possible sediment-water interactions relevant to the release of
toxic metals.
Geological settings of the study area
Fig. 1. Geological settings of Taiwan showing convergent plate
boundary between the Eurasian Plate and the Philippine Sea Plate
(source: Central geological survey, MOEA, Taiwan).
Previous studies reported high concentrations of Na+,
Cl , and SO4 2− in hot spring fluids, however fluid
chemical compositions are mainly dependant on the
source components and sediment-water interactions deep
underground.[13–16] In Taiwan, many studies have reported
the geochemical characterization of mud volcano fluids,[17–19] while a few studies have linked mud volcanic
activity and the distribution of arsenic (As) and other
−
Taiwan is located in the zone of active continental deformation within the convergent plate boundary between the
Eurasian Plate and the Philippine Sea Plate (Figs. 1 and
2). The plate interaction is marked by the collision of
the Luzon volcanic arc and the Asiatic continental margin. The Cenozoic geology of Taiwan is closely related
to the Ryukyu and Luzon arc-trench systems. The geologic structure of the area can be divided into 5 major tectonic units. From west to east, the units are Coastal Plain,
Western Foothills, Hsuehshan Range, Central Range,
′
and the Coastal Range. Kuan-Tzu-Ling (23◦ 20 31.0′′N;
◦ ′
′′
◦ ′
′′
◦ ′
120 29 48.9 E), Chung-Lun (23 22 34.2 N; 120 33 04.7′′E)
′
′
and Bao-Lai (23◦ 05 18.4′′N; 120◦ 42 43.6′′E) are the bestknown hot springs in southern Taiwan (Figs. 3a–c).
Fig. 2. Map of the study area showing location of Chung-Lun, Kuan-Tzu-Ling and Bao-Lai hot springs in southern Taiwan. Location
of Chianan plain, Tainan and Kaohsiung city are also shown in the map. Inset picture shows a map of Taiwan and the corresponding
study area location.
Biochemical characteristics of hot springs in southern Taiwain
1209
HI-9143 Microprocessor Dissolved Oxygen Meter
(HANNA, Taiwan). Alkalinity was determined by the
titrimetry method in the field.
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
Laboratory chemical analysis
The samples were filtered in the laboratory using 0.45 µm
filter paper. The filtered samples were measured for cation
(Na+, K+, Mg2+ and Ca2+) and anion (Cl−, NO2− and
SO4 2−) concentrations using an Ion Chromatograph (IC)
(Dionex, CA, USA). Trace element concentrations were
measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Hewlett-Packard 4500, YamanashiKen, Japan). Accuracy was checked in each analytical batch
by measuring certified reference material ‘Trace Metals in
Drinking Water’ (TMDW; Lot # 623609) from High Purity
Standards (HPS, USA), and was found to be within ±5%
of the certified values (accuracy: ±5% and precision: ±2%).
Fig. 3. Major hot springs in southern Taiwan: (a) Kuan-TzuLing, (b) Chung-Lun and (c) Bao-Lai; (d) Inoculation of hot
spring water for bacterial growth in the field (color figure available
online).
Materials and methods
Sampling collection and preservation
The fluid and mud sediment samples were collected in
dark sterilized bottles (500 mL in triplicates) from different eruption points of the hot springs. In the laboratory, the samples were flushed with N2 and stored under
anaerobic conditions. The collected fluid and mud sediment samples were later analyzed for chemical constituents
detailed in the sections below. Fluid samples for microbiological studies were inoculated into serum bottle containing sulfate reducing media (Dipotassium phosphate 0.50
g/L; Peptic digest of animal tissue 2.00 g/L; Beef extract
1.00 g/L; Sodium sulphate 1.50 g/L; Magnesium sulphate
heptahydrate 2.00 g/L; Calcium chloride 0.10 g/L; Ferric ammonium sulphate 0.392 g/L; Sodium ascorbate 0.10
g/L; Sodium lactate 3.50 and Final pH 7.5 ± 0 at 25◦ C; in
the field under sterile conditions). In the laboratory such
samples were preserved anaerobically for later microbial
studies.
Extraction of humic substances and study of the functional
groups
Humic substances (HS) were extracted from the sediments and analyzed for fluorescence characteristics (e.g.,
functional groups and fluorescence sensitivity). Sediment
samples were air-dried and passed through 0.2 mm sieve.
After homogenization, an aliquot of samples were dissolved in a volume (1:10 ratio) of 0.1M NaOH under N2 -H2
(95:5, V/V) atmosphere in a glove box, sealed, and agitated
on a rotary shaker at 30◦ C for 24 h. Then the solution was
centrifuged at 5000 g for 15 min under N2 to remove impurities.
The supernatant was collected and acidified with 6N
HCl. The HA was precipitated with HCl and allowed to
stand for 16–20 h, and centrifuged at 1500 g for 10 min.
The precipitated HA was treated three times with HCl-HF
by shaking overnight to remove impurities and freeze dried
to get HA powder sample. The extracted humic acid fractions were analyzed by Fourier Transform Infrared Spectrophotometer (FTIR).[22] The data were interpreted using
KnowItAll and Spectrum software to analyze functional
groups and possible chemical components of humic acid.
Field measurement of environmental parameters
Fluorescence intensity of dissolved humic substances in the
fluids
Electrical conductivity (EC), total dissolved solids (TDS),
dissolved oxygen (DO), temperature, salinity, pH, Eh and
alkalinity were measured in the field. A portable conductivity meter was used to measure EC, salinity and
TDS (Suntex; WTW, LF320, Germany). pH, Eh and
temperature were measured using a multimeter (SP701,
Suntex, Kaohsiung, Taiwan). DO was measured by the
Fluids were filtered through 0.45 µm filter paper. The fluorescence intensity of dissolved humic substances (HS) were
detected by a fluorescence spectrophotometer (Model F4500, Hitachi, Japan), and the concentration of HS was
measured by quinic acid standard curve.[23] The excitation
and absorption wavelengths for the detection of the fluorescence intensity were 347 and 445 nm.[23]
1210
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
Isolation and physiological characteristics of the bacterial
isolates
The geothermal fluids were used to cultivate bacteria using Sulfate-Reducing Bacteria (SRB) medium (Hi-Media,
Mumbai, India) in an anaerobic environment,[24] followed by bacterial isolation in Hi-Media agar plate using the dilution plate method described in Dubey and
Maheshwari.[25]
The Genomic DNA extraction kit (Viogene, Taipei,
Taiwan) was used after the bacteria were sufficiently incubated (dominant two bacteria: Acc. No. GU329907 and
GU329908). The culture medium was centrifuged at 7,500 g
for 10 min and the supernatant was removed. The pellet was
extracted with 200 µL lysozyme reaction solution (20 mM
Tris-HCl, pH = 8.0; 2 mm EDTA; 20 mg/mL lysozyme).
The eppendorf tubes were incubated horizontally at 37◦ C
for 30 min. Then 20 µL proteinase K and 200 µL EX Buffer
were added and vortexed for 20 s with further incubation
at 60◦ C for 30 min. Then, 210 µL isopropanol was added
to each Eppendorf tube and mixed thoroughly. All fluids
were then transferred to each genomic DNA Mini Column
with Eppendorf and centrifuged at 7,500 g for 10 min. After removal of the supernatant, the pellet was treated two
times with 500 µL WS Buffer and centrifuged at 8,000 g
for 2 min. The genomic DNA Mini Column was dried at
room temperature and then dissolved in 50 µL TE Buffer
(10 mM Tris Cl, 1 mM EDTA, pH 8.0).
Maity et al.
The DNA sequences were reproduced with forward
and reverse primers, and the 16S 27F (forward): 5′ –A
GAGTTTGATC CTGGCTC AG-3′ (genomic positions
8–27, Escherichia coli numbering) and 16S 1492R (reverse):
5′ –GGTTAC CTTGTTACGACTT-3′ (genomic positions
1488–1511, Escherichia coli numbering) were identified for
this study.[26] The universal forward and reverse primers
were synthesized by Mission Biotech Co. Ltd. (Taipei,
Taiwan). The gene sequencing was made from the PCR
product through a gene sequencer (ABI 3730 XL DNA
Analyzer, Applied Biosystems, CA, USA). These sequence
fractures (∼1500 bp) were doing blast in GeneBank of
NCBI, to compare the difference for nucleotide sequences
of the bacteria. 16S rDNA were aligned with the CLUSTAL
X 1.81 program.[27] Neighbor-joining (NJ) analysis[28] based
on two-parameter distance was performed using the software MEGA 2.0.[29] Confidence of the clade reconstruction
was tested by bootstrapping with 1000 replicates using unweighted characters.[30]
Estimation of sulfate and arsenate reduction
Bacterial
isolates,
Desulfovibrio
psychrotolerans
(GU329907) and Clostridium sulfidigenes (GU329908)
were cultured under anaerobic condition with a initial
SO4 2− concentration of 150 mg/L at 28◦ C for 20 days.
The concentrations of SO4 2− and S2− were determined
Fig. 4. Durov diagram showing the composition of chemical components along with TDS and pH in geothermal water (color figure
available online).
1211
Biochemical characteristics of hot springs in southern Taiwain
Table 1. Field measured environmental parameters of geothermal fluids in southern Taiwan hot springs.
Field measured environmental parameters
Sampling sites
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
Bao-Lai
Kuan-Tzu-Ling
Chung-Lun
pH
7.01 ± 0.02
7.41 ± 0.02
8.00 ± 0.02
Eh (mV)
35
−176
−140
Temperature
(◦ C)
Electrical
conductivity
(mS/cm)
Salinity ()
DO (mg/L)
TDS†
(mg/L)
62.0 ± 0.7
82.2 ± 1.2
50.0 ± 0.4
2.15 ± 0.03
13.6 ± 0.09
36.3 ± 1.20
1.0 ± 0.02
8.3 ± 0.03
23.0 ± 0.05
0.82 ± 0.003
0.47 ± 0.003
0.32 ± 0.002
1,077 ± 8.2
13,980 ± 11
64,125 ± 12
†
The data presented as statistical mean and standard error (n = 6).
†
DO: Dissolve Oxygen; TDS: Total Dissolved Solids.
by Ion Chromatograph and Microprocessor-Controlled
Photometer-PC MultiDirect. The same bacterial isolates
were also cultured under anaerobic condition with
an initial As(V) concentration of 2000 µg/L at 28◦ C
for 15 days. The culture was conducted with modified
carbon-free media (NaCl, 2.00 g; MgCl2 .6H2 O, 0.40 g;
CaCl2 .2H2 O, 0.10 g; Na2 SO4 , 4.00 g; NH4 Cl, 0.25 g;
KH2 PO4 , 0.20 g; KCl, 0.50 g; selenite solution, 1.00 ml;
trace elements, 1.00 ml). The concentration of As(V)
and As(III) was measured by using the Millenium
Excallibur Fluorecence Atomic Analyser (HG-AFS)
(PS Analytical Ltd, Kent, UK) coupled with HPLC.
The reduction of sulfate and arsenate were measured at
regular intervals to understand the activity of bacterial
isolates.
Statistical analysis
Data were presented as minimum, maximum, mean and
standard deviation. The correlation statistics and level of
statistical significance were analyzed using the statistical
Fig. 5. Piper diagram showing ionic characteristics of the fluids collected from different hot springs of southern Taiwan (color figure
available online).
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
1212
Maity et al.
Fig. 6. Schoeller diagram showing variation of major ionic constituents in different geothermal waters (color figure available online).
software SPSS 13.0.[31] The p value less than 0.05 was
considered to indicate statistical significance. Linear
regression was used to find correlation between different
biogeochemical parameters.
Quality control
Analytical accuracy was checked with reference material
and precision was ensured through repeated measurements.
For total As analysis, standard reference material (SRM
3103a, NIST, USA) was used. The detection limit of total
As was 0.1 µg/L. The coefficient of variation (CV) was
calculated to test the reliability of the data and was found
to be <5% in all the replicates.
Results and discussion
Environmental parameters
The environmental parameters (e.g., temperature, pH,
salinity, EC, Eh, TDS and DO) were measured at in-situ
conditions (Table 1). The temperature of the KTL hot
spring is 82.2 ± 1.2◦ C; whereas 62.0 ± 0.7◦ C and 50.0 ±
0.4◦ C for BL and CL hot springs, respectively. The pH
of the CL fluids is higher (8.00 ± 0.02) in comparison to
BL (7.41 ± 0.02) and KTL (7.01 ± 0.02) fluids (Fig. 4).
The TDS of CL is much higher compared to the BL and
KTL fluid (Fig. 4). The salinity of the CL (23.0 ± 0.05
) is higher than that of the KTL and BL fluids. The Eh
values of KTL (Eh value: −396 mV) and CL (Eh value:
−360 mV) fluids are low compared with BL (Eh value:
−185 mV), suggesting reducing nature.
Table 2. Contents of the major oxides (%) in mud sediments collected from geothermal hot springs in southern Taiwan.
Major oxides (in wt%)
Sampling sites
SiO2
TiO2
Al2 O3
Fe2 O3
MnO
MgO
CaO
Na2 O
K2 O
P2 O5
LOI
Bao-Lai
Kuan-Tzu-Ling
Chung-Lun
62.98
58.65
55.13
0.92
1.02
1.45
15.49
16.75
18.25
6.12
7.50
8.26
0.08
0.08
0.58
2.31
2.61
3.15
2.28
2.75
3.85
1.66
1.49
1.85
2.86
3.05
4.25
0.15
0.17
0.19
5.15
5.93
3.04
1213
Biochemical characteristics of hot springs in southern Taiwain
Table 3. Trace element concentrations in geothermal fluids and mud sediments collected from southern Taiwan.
Trace elements (for fluids: µg/L, and for solids: µg/kg)
Sampling sites
KTL (S)†
KTL (L)†
CL (L)
BL (L)
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
†
As
Ba
Cd
Co
Cr
Cu
Fe
Mo
Mn
Ni
Rb
Se
Sr
V
Zn
121
410
91
4.03
169
354
6.99
8.18
BDL†
BDL
BDL
BDL
ND
BDL
BDL
BDL
77.2
BDL
BDL
BDL
BDL
104
0.24
0.23
2,090
17.1
36.0
43.4
BDL
BDL
BDL
BDL
6,152
BDL
3.69
2.68
11.1
BDL
0.06
0.07
BDL
95.6
22.9
27.2
BDL
2.05
1.05
3.05
73.1
530
22.1
26.0
124
BDL
0.08
0.07
22.3
BDL
0.23
0.24
L: Liquid; S: Sediment; BDL: Below Detection Limit.
Geochemistry of the geothermal fluids
and sediment
The chemical characteristics of the geothermal fluids are
shown in a Piper diagram (Fig. 5). Na-Cl and K-Cl are the
major water types of geothermal fluids. Specifically KTL
Table 4. Functional groups of humic substances in geothermal
fluids: (a) Kuan-Tzu-Ling, (b) Chung-Lun and (c) Bao-Lai, in
Southern Taiwan.
Classification
Group
(a)
Alkanes
Alcohols
Amides-I
Amides-II
Amides-III
Amines
Aromatic
Ethers-IR
Halogens
Phosphorus
CCCH CHCC
Ph–CHR–OH
–CO–NH–C
–CO–NH–C
–CO–NH–C
CH2–NH2
o-Disubst
Ph–O–C
C-F
P O
Sulfur
(b)
Amines
Amides-I
Amides-II
Amides-III
Ureas
Urethanes
Phosphorus
Sulfur
(c)
Alkanes
Alcohols
Amides-I
Amides-II
Amides-III
Amines
Aromatic
Sulfur
Bond
Range (cm−1)
CC
O–H
C O
CNH
CNH
NH2
Ring
C–O–C
C-F
P O
P-O-R
S O
2849–2917
3400–3200
1680–1630
1570–1515
1305–1200
1650–1590
1625–1590
1050–1010
1400–1000
1300–1250
1050–970
1090–990
R-SOOH
N-H
C O
CNH
CNH
NH2 ,
C O
N-C-N
C-N, C-C
P O
P-O-R
S O
3500–3300
1680–1630
1570–1515
1305–1200
3440–3400
1680–1635
1190–1140
1265–1200
1300–1250
1050–970
1090–990
CCCH CHCC
Ph–CHR–OH
–CO–NH–C
–CO–NH–C
–CO–NH–C
CH2–NH2
o-Disubst
R-SOOH
CC
O–H
C O
CNH
CNH
NH2
Ring
S O
2849–2917
3400–3200
1680–1630
1570–1515
1305–1200
1650–1590
1625–1590
1090–990
R-SOOH
CH2-NH-CH2
–CO–NH–C
–CO–NH–C
–CO–NH–C
R-NH-CO-NH2
NH2COO-R
P O
and CL geothermal fluids are of mixed composition with
Cl− and HCO3 − as the dominant anions, whereas BL hot
spring fluids mainly consist of Na-HCO3 and K-HCO3 type
fluids (Fig. 6). Salinity data suggest that the CL geothermal
fluids might originate from seawater. The major oxides of
the geothermal sediments are shown in Table 2. SiO2 is generally high in BL while Al2 O3 and Fe2 O3 are enriched in CL
and KTL. High contents of Al2 O3 and Fe2 O3 in the fluids
are likely due to enrichment in clay minerals and feldspars.
Trace elemental composition of geothermal fluids
and sediments
The geothermal fluids (except for BL hot spring) contain
As concentrations above the WHO recommended drinking water safe limit of 10 µg/L. The KTL hot spring contains the highest As concentration (410 µg/L) compared
with other hot springs (91 and 4.03 µg/L, for CL and
BL, respectively) (Table 3). Both KTL and CL hot springs
are situated close to the Chianan plain of southwestern
Taiwan, where groundwater contains considerable amounts
of As.[20]
Trace elements such as Ba (range: 7 and 354 µg/L), Rb
(range: 23 and 96 µg/L), Se (range: 1.05 and 3.05 µg/L)
and Sr (range: 22 and 530 µg/L) are continuously sourced
from hot springs and thereby increasing toxic elemental
burden to the environment (Table 3). The results also exhibited high concentrations of Cr, Fe, Mn, Ni, Sr, V and
Zn in the sediments, while low concentrations are observed
in the fluid phase.
Functional group of humic substances
Humic substances are dark brown in color and are the
major constituents of soil organic matter that contribute
to soil chemical and physical quality. Humic substances
have a special capacity to bind metals and control
mobility of metal(oid)s. The FTIR spectra shows the
presence of possible functional groups of aromatics,
secondary amines, ureas and proteins such as amide-I,
amide-II, and fatty acids (Table 4). The OH stretching vibration and possibly NH stretching vibration
(3300–3500 cm−1) are from primary amines, CH stretching
vibration (3000–3100 cm−1), -C O strong vibration
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
1214
Maity et al.
Fig. 7. Correlation between dissolved humic substances and As in fluids (a), and As in sediments (b).
(1680–1740 cm−1), -C O non-cyclic saturated asymmetricstretching vibration (1800–1850 cm−1), -C O non-cyclic
conjugated asymmetric-stretching vibration (1770–1780
cm−1), C-O-C strong asymmetric-stretching vibration from ether, (1060–1150 cm−1), and C-F very
strong stretching vibration (1000–14000 cm−1) from
halogens.
The dominant functional groups that contribute to surface charge and reactivity of humic substances are phenolic
and carboxylic groups.[32] Humic substances may chelate
divalent cations such as Mg2+, Ca2+, and Fe2+. Our study
shows the presence of possible functional groups of primary
and secondary amines, carboxyl, phenol group, ureas, and
urethanesm, which suggests the ability of humic substances
to form chelate complexes, thereby increasing the availability of many cations in the geothermal fluids. We observed a
strong positive correlation with dissolved humic substances
and As in fluids (Fig. 7a).
However, no such correlation is observed between dissolved humic substances and As in sediments (Fig. 7b). This
suggests that As might have released fluids together with
dissolution of humic substances under strongly reducing
Fig. 8. Phylogenic relationship between bacterial isolates and reference strains of Chung-Lun, Kuan-Tzu-Ling and Bao-Lai hot
springs. The calculation of evolutionary distance and classification of phylogenetic relationship were determined using Jukes-Cantor
distance and neighbour-joining algorithm. The scale bar represents 0.05 fixed-point changes per nucleotide position.
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
Biochemical characteristics of hot springs in southern Taiwain
1215
Fig. 9. Sulphate reduction in presence of two bacterial isolates: Desulfovibrio psychrotolerans (GU329907) and Clostridium sulfidigenes
(GU329908) (color figure available online).
Fig. 10. Arsenic reduction in presence of two bacterial isolates: Desulfovibrio psychrotolerans (GU329907) and Clostridium sulfidigenes
(GU329908) (color figure available online).
1216
conditions. Previous studies showed that humic substances
have the ability to reduce As(V) to more mobile As(III)
species.[33] Organic matter in general can influence solubility of As due to competitive adsorption, complexation, and
redox reactions.[34]
Maity et al.
Acknowledgment
The authors wish to thank National Science Council,
Taiwan for financial support.
References
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
Bacterial signature
The isolated strain from KTL hot spring is classified as
the Bacillus genera. The nucleotide similarities between
the isolated strains from KTL: B. pocheonensis (Accession
no: FJ009384); B. drentensis (Accession no: FJ009411); B.
soli strain G8 (Accession no: FJ009379) and B. arbutinivorans (Accession no: FJ380988) are 99.0, 98.0, 98.0, and
98.0 (Fig. 8). Based on this result, bacterial isolates from
KTL are assigned as B. pocheonensis. Similarly, the isolated
strains from BL are assigned as Clostridium sulfidigenes,
while the isolated strains from CL hot spring are assigned
as Desulfovibrio psychrotolerans.
Hot springs are rich in different types of reducing bacteria (such as Clostridium sulfidigenes and Desulfovibrio psychrotolerans) which mediate the release of redox sensitive
elements (e.g., As) to hot spring fluids. In the laboratory, we
studied the activity of such bacterial isolates in microcosm.
Our results demonstrate that the bacterial isolates from hot
spring fluids are able to reduce both sulfate and arsenate
(Figs. 9 and 10), implying the release of mobile As(III) into
the hot spring fluids under reducing conditions. However,
precipitation of secondary sulfides (e.g., mackinawite) due
to sulfate reduction may remove some of the dissolved As
from the hot spring fluids (Fig. 10).[35,36]
Conclusions
The high concentrations of Na+ and Cl− in the geothermal
fluids suggest a possible marine origin. The distribution
of trace elements in the fluids, especially As, suggests that
redox processes in the fluids are responsible for release of
As. The FTIR spectra of the extracted humic fractions
of the sediments showed the presence of possible functional group of aromatics, secondary amines, ureas and
protein fraction. This suggests the ability of humic substances to form chelating complex and thereby increasing
the mobility of many cations, including As. The bacterial
strains isolated from the geothermal fluids can reduce sulfate and arsenate in the fluids under anoxic conditions.
We infer that the geothermal fluids likely contribute to
groundwater As in the downstream alluvial plain. However, detailed trace element and isotope chemistry will
be required to determine the exact role of hot springs
in the genesis of arseniferous aquifers in the Chianan
plain.
[1] Ballantyne, J.M.; Moore, J. Arsenic geochemistry in geothermal
systems. Geochim. Cosmochim. Acta 1988, 52, 475–483.
[2] Christensen, O.D.; Capuano, R.A.; Moore, J.N. Trace-element distribution in an active hydrothermal system, Roosevelt hot springs
thermal area, Utah. J. Volcanol. Geothermal Res. 1983, 16, 99–
129.
[3] Lazareva, E.V.; Bryanskaya, A.V.; Zhmodik, S.M.; Kolmogorov,
Y.P.; Pestunova, O.P.; Barkhutova, D.D.; Zolotarev, K.V.;
Shaporenko, A.D. Elements redistribution between organic and
mineral parts of microbial mats: SR-XRF research (Baikal Rift
Zone). Nuclear Instr. Meth. Phys. Res. Sect. A: Accelerators, Spectrometers, Detectors Asso. Equip. 2009, 603, 137–140.
[4] Möller, P.; Dulski, P.; Savascin, Y.; Conrad, M. Rare earth elements,
yttrium and Pb isotope ratios in thermal spring and well waters of
West Anatolia, Turkey: a hydrochemical study of their origin. Chem.
Geol. 2004, 206, 97–118.
[5] Horváth, A.; Bohus, L.O.; Urbani, F.; Marx, G.; Piróth, A.;
Greaves, E.D. Radon concentrations in hot spring waters in northern Venezuela. J. Env. Radioacti. 2000, 47, 127–133.
[6] Sompong, U.; Hawkins, P.R.; Besley, C.; Peerapornpisal, Y. The
distribution of cyanobacteria across physical and chemical gradients
in hot springs in northern Thailand. FEMS Microbiology Ecol.
2005, 52, 365–376.
[7] Sanada, T.; Takamatsu, N.; Yoshiike, Y. Geochemical interpretation of long-term variations in rare earth element concentrations
in acidic hot spring waters from the Tamagawa geothermal area,
Japan. Geothermics 2006, 35, 141–155.
[8] Momoshima, N.; Nita, J.; Maeda, Y.; Sugihara, S.; Shinno, I.;
Matsuoka, N.; Huang, C.W. Chemical composition and radioactivity in hokutolite (plumbian barite) collected at Peito hot spring,
Taiwan. J. Env. Radioacti. 1997, 37, 85–99.
[9] Jang, C.S. Applying scores of multivariate statistical analyses to
characterize relationships between hydrochemical properties and
geological origins of springs in Taiwan. J. Geochem. Explor. 2010,
105, 11–18.
[10] Alfaro, C.; Wallace, M. Origin and classification of springs and
historical review with current applications. Env. Geol. 1994, 24,
112–124.
[11] Shih, T.T. A survey of the active mud volcanoes in Taiwan and a
study of their types and the character of the mud. Petrol. Geol.
Taiwan 1967, 5, 259–311.
[12] Wang, S.; Shu, M.; Yang, C. Morphological study of mud volcanoes
on land in Taiwan. J. Nat. Taiwan Museum 1988, 31, 31–49.
[13] Davisson, M.L.; Avisson, M.L.; Presser, T.S.; Criss, R.E. Geochemistry of tectonically expelled fluids from the northern coast ranges,
Rumsey-Hills, California, USA. Geochim. Cosmochim. Acta 1994,
58, 1687–1699.
[14] Minissale, A.; Magro, G.; Vaselli, O.; Verrucchi, C.; Perticone, I.
Geochemistry of water and gas discharges from the Mt. Amiata
silicic complex and surrounding areas (central Italy). J. Volcanol.
Geotherm. Res. 1997, 79, 223–251.
[15] Mariner, R.H.; Evans, W.C.; Presser, T.S.; White, L.D. Excess nitrogen in selected thermal and mineral springs of the Cascade Range
in northern California, Oregon, and Washington: sedimentary or
volcanic in origin? J. Volcanol. Geotherm. Res. 2003, 121, 99–114.
[16] Afşin, M.; Kuşcu, I.; Elhatip, H.; Dirik, K. Hydrogeochemical properties of CO2 -rich thermal-mineral waters in
Kayseri (Central Anatolia), Turkey. Environ. Geol. 2006, 50, 24–36.
Downloaded by [National Chung Cheng University], [Jyoti Prakash Maity] at 23:44 31 August 2011
Biochemical characteristics of hot springs in southern Taiwain
[17] Gieskes, J.M.; You, C.-F.; Lee, T.; Yui, T.-F.; Chen, H.-W. Hydrogeochemistry of mud volcanoes in Taiwan. Acta Geol. Taiwan 1992,
30, 79–88.
[18] Yang, T.F.; Yeh, G.H.; Fu, C.C.; Wang, C.C.; Lan, T.F.; Lee, H.F.;
Chen, C.H.; Walia, V.; Sung, Q.C. Composition and exhalation flux
of gases from mud volcanoes in Taiwan. Environ. Geol. 2004, 46(8),
1003–1011.
[19] You, C.-F.; Gieskes, J.M.; Lee, T.; Yui, T.-F.; Chen, H.-W. Geochemistry of mud volcano fluids in the Taiwan accretionary prism. Appl.
Geochem. 2004, 19, 695–707.
[20] Nath, B.; Jean, J.-S.; Lee, M.-K.; Yang, H.-J.; Liu, C.-C. Geochemistry of high arsenic groundwater in Chia-Nan plain, Southwestern
Taiwan: possible sources and reactive transport of arsenic. J. Contam. Hydrol. 2008, 99, 85–96.
[21] Liu, C.C.; Jean, J.S.; Nath, B.; Lee, M.K.; Hor, L.I.; Lin, K.H.;
Maity, J.P. Geochemical characteristics of the fluids and muds
from two southern Taiwan mud volcanoes: Implications for watersediments interaction and groundwater arsenic enrichment. Appl.
Geochem. 2009, 24, 1793–1802.
[22] Muñoz, M.A.; Carmen, C.; Manuel, B. FTIR and fluorescence studies on the ground and excited state hydrogen-bonding interactions
between 1-methylindole and water in water–triethylamine mixtures.
Chem. Phys. 2007, 335, 43–48.
[23] ROC EPA. Standard Method of Water Analysis, NIEA W940.50T,
1994.
[24] George, J.; Purushothaman, C.S.; Shouche, Y.S. Isolation and characterization of sulphate-reducing bacteria Desulfovibrio vulgaris
from Vajrechwari thermal springs in Maharashtra, India. World
J. Microbiol. Biotech. 2008, 24, 681–685.
[25] Dubey, R.C.; Maheshwari, D.K. Practical Microbiology. S. Chand
and Company Ltd., New Delhi, India, 2005.
[26] Goorissen, H.P.; Boschker, H.T.S.; Stams, A.J.M.; Hansen, T.A.
Isolation of thermophilic Desulfotomaculum strains with methanol
and sulfite from solfataric mud pools, and characterization of Desul-
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
1217
fotomaculum solfataricum sp. nov. Int. J. Syst. Evol. Microbiol. 2003,
53, 1223–1229.
Thompson, J.D.; Gibson, T.J.; Plewniak, F.; Jeanmougin, F.;
Higgins, D.G. The Clustal X windows interface: flexible strategies
for multiple sequence alignment aided by quality analysis tools.
Nucleic Acid Res. 1997, 24, 4876–4882.
Kimura, M. A simple method for estimating evolutionary rates of
base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980, 16, 111–120.
Kumar, S.K.; Tamura, I.B.; Jakobsen; Nei, M. MEGA2: molecular
evolutionary genetics analysis software. Bioinformatics 2001, 17,
1244–1245.
Felsenstein, J. Confidence limits on phylogenies: an approach using
the bootstrap. Evolution 1985, 39, 783–791.
Chen, B.; Liu, H. Relationships between phytoplankton growth
and cell size in surface oceans: Interactive effects of temperature,
nutrients, and grazing. Limnol. Oceanogr. 2010, 55, 965–972.
Stevenson, F.J. Humus Chemistry: Genesis, Composition, Reactions.
Wiley and Sons, New York, 1994.
Palmer, N.E.; Freudenthal, J.H.; von Wandruszka, R. Reduction
of arsenates by humic materials. Environ. Chem. 2006, 3, 131–
136.
Wang S.; Mulligan, C.N. Effect of natural organic matter on arsenic release from soils and sediments into groundwater. Environ.
Geochem. Health 2006, 28, 197–214.
Ahmed, S.; Alexander, S. Anaerobic and aerobic degradation
of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1 and role of three other coisolates in a
mixed bacterial consortium. Appl. Env. Microbiol. 2008, 74, 3434–
3443.
Bonneville, S.; Behrends, T.; van Cappellen, P.; Hyacinthe, C.;
Röling, W.F.M. Reduction of Fe(III) colloids by Shewanella putrefaciens: a kinetic model. Geochim. Cosmochim. Acta 2006, 70,
5842–5854.