International Biodeterioration & Biodegradation 136 (2019) 55–62

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Arsenite oxidation by a facultative chemolithotrophic Delftia spp. BAs29 for T

its potential application in groundwater arsenic bioremediation
Rimi Biswasa, Vivekanand Vivekananda, Anima Sahaa, Ashok Ghoshb, Angana Sarkara,∗
a
Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha, 769008, India
b
Mahavir Cancer Institute and Research Center, Patna, India

A R T I C LE I N FO A B S T R A C T

Keywords: A superior arsenite [As (III)] oxidizing strain was isolated by enrichment technique from a shallow aquifer of
Arsenic Bhojpur district, Bihar, India. Along with high As (III) and arsenate [As (V)] resistance [up to 70 mM of As (III)
Arsenite oxidizing bacteria and 1000 mM of As (V), respectively], it showed high resistance to multi-metals and a superior ability to utilize a
Groundwater wide range of carbon sources. The gram-negative rod-shaped strain of Delftia spp. BAs29 was characterized
Bioremediation
thoroughly for its As (III) oxidation ability. The strain showed mixed growth associated, facultative chemo-
Bio-sorbents
Bio-transformation
lithotrophic As (III) oxidation process, releasing-67 kJ/reaction of free energy in an exergonic reaction. Testing
of cost-effective and natural bio-sorbents showed efficient As (V) removal from the contaminated water. Moringa
oleifera showed the maximum As (V) removal capacity of 57.89% among the tested bio-sorbents. This study
revealed a two-step approach for arsenic removal with 98.77% efficiency from groundwater using an indigenous
As (III) oxidizing strain combined with a natural bio-sorbent.

1. Introduction
Increased level of arsenic (As) is a threat to mankind, affecting
millions of people every year worldwide (Paul et al., 2015). The middle
Gangetic plain, including several districts of Bihar and West Bengal,
seem to be the worst affected areas, affecting around 40 million people.
In this region, total As concentration is much greater than the pre-
scribed limit (0.01 mg/l) by WHO (Chakraborti et al., 2003). Long-term
exposure to toxic levels of As leads to skin cancer, loss of appetite, skin
itching and cardiac problems (Banerjee et al., 2011). Geochemical
weathering of rocks, anthropogenic activities and volcanic emissions
are the primary causes of groundwater As contamination. Geo-micro-
bial ecotoxicology is gaining prime importance in the field of en-
vironmental science and research (Gu et al., 2013). Microbial bior-
emediation of As is quite effective and depends on the intrinsic action of
microbial activity to transform, volatize, reduce, immobilize or mobi-
lize As through redox reactions, sorption, complexation, and bio-
methylation (Dai et al., 2016; Gu, 2018).
As mainly exists in four different valences in the natural environ-
ment; elemental arsenic (0), arsenine (-III), arsenite (III) and arsenate
(V). Among these, As (III) and As (V) are the two most abundant forms
in the environment; with As (III) being the more toxic and more mobile
form than its counterpart (Aitio and Becking, 2001). The removal of As
(III) from contaminated water is difficult due to its high magnitude of
solubility, while As (V) can be easily removed by some conventional
physicochemical methods like filtration, adsorption, reverse osmosis,
membrane filtration, and coagulation etc. (Bahar et al., 2012). A two-
step approach can be adapted to remove total As, wherein the first step
involved is the oxidation of more toxic As (III) to As (V) and the next
step is associated with the use of adsorbents for the removal of oxidized
As (V). The oxidation step requires toxic chemical oxidants like ozone,
chlorine or hydrogen peroxide which either produce hazardous by-
products or are expensive. Alternatively, microbes in their metabolic
processes use As (III) as an efficient energy source, aiding the process of
reducing the groundwater toxicity (Kamaluddin et al., 2003). Many
potential As (III) oxidizing bacteria including Bacillus cereus, Alcaligenes
faecalis, Achromobacter xylosoxidans, Rhizobium spp., Pseudomonas spp.,
Agrobacterium tumifaciens, Escherichia coli, Clostridium spp., Rumino-
coccaceae spp., Klebsiella oxytoca and Acinetobacter calcoaceticus have
been reported to withstand high concentrations of As and can sub-
stantially aid in the bioremediation of As from groundwater (Sarkar
et al., 2014; Paul et al., 2015; Tripti and Shardendu, 2017; Liu et al.,
2018; Dai et al., 2016). Arsenite oxidizers may be either heterotrophic
i.e. they oxidize As (III) only as a mode of detoxification while the
chemolithotrophic As (III) oxidizers gain energy during this process
(Oremland and Stolz, 2005). In prokaryotes, As (III) oxidation is

∗
Corresponding author.
E-mail addresses: rimi.biswas12@gmail.com (R. Biswas), vk7maurya@gmail.com (V. Vivekanand), animasaha.179@gmail.com (A. Saha),
ghosh51@hotmail.com (A. Ghosh), sarkara@nitrkl.ac.in, sarkar.angana@gmail.com (A. Sarkar).

https://doi.org/10.1016/j.ibiod.2018.10.006
Received 31 May 2018; Received in revised form 12 October 2018; Accepted 12 October 2018
0964-8305/ © 2018 Published by Elsevier Ltd.
R. Biswas et al.
controlled by a periplasmic As (III) oxidase enzyme (Andreoni et al.,
2012). With respect to highly expensive and conventional As removal
technologies which are ineffective for the removal of low As con-
centration, extensive technological developments that are highly se-
lective and economically competitive are strongly in demand. Since
natural microorganisms have intrinsic abilities to detoxify toxic As
species, it is of immense importance to identify and characterize such
organisms and their As detoxification mechanisms.
Solid adsorbents are more popular in As (V) adsorption due to the
economic and technical limitations for the use of standard flocculation
and precipitation methods. Activated -carbon, -alumina and -bauxite
are some of the adsorbents used for the study of As removal from
aqueous solutions (Lim et al., 2014). Usage of natural adsorbents such
as leaves, sand, red mud etc., are gaining momentum, aiding the As
removal process. They are highly impactful as they do not produce any
hazardous by-products and are cost effective.
Although a thorough geochemical characterization has been carried
out in the middle Gangetic plains of Bihar (Particularly, Bhojpur dis-
trict), the reports on the characterization of As transforming bacteria is
limited. As per our knowledge, this is the first time a thorough research
is elucidating a two-step approach of As removal, where a test strain is
exploited to transform more mobile and toxic As (III) to its less toxic
and mobile form As (V), following its adsorption on eco-friendly and
inexpensive natural bio-sorbents. This approach could be highly effi-
cient in the bioremediation of As, aiding significant contribution in the
process of total As removal from any As contaminated groundwater.
2. Materials and methods
2.1. Collection of samples and storage
The water samples were collected in the early winter season from
two different As contaminated sites of Dubechaapra (25.6462670N,
84.6742670E) and Gajiapur villages (25.6869780N, 84.6366080E) of
Bhojpur district in Bihar lying in the middle Gangetic plains (Fig. 1).
The inherent temperature of the groundwater samples was 25 °C at a pH
Fig. 1. Arsenic contaminated groundwater sample collection sites from Bhojpur district, Bihar, India.
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International Biodeterioration & Biodegradation 136 (2019) 55–62
of 7.1. For each sample, two different sterile polypropylene bottles were
used. In one sample, nitric acid was added to avert the metal from
microbial degradation, adsorption and precipitation while the second
sample was kept at 4 °C on ice for further microbial analysis.
2.2. Enrichment and isolation of As resistant bacteria
Isolation of As resistant bacteria was done directly from the As
contaminated water as well as enriching it with As (III) (10 mM) and As
(V) (1 mM) for 7 days, 120 rpm at 30 °C (Sarkar et al., 2013). The initial
growth and enrichment of the strain was done in Reasoner's 2A (R2A)
agar medium under aerobic conditions at 30 °C. Throughout the in-
cubation period, counting of the number of bacterial colony forming
units (CFU's) was done at definite time intervals. A morphologically
distinct isolate was selected based on its highest As resistance for fur-
ther characterization. The isolate was purified by sub-culturing re-
peatedly in a suitable medium and stored at 80 °C a glycerol stock.
2.3. Arsenic transformation screening assay
Determination of As (III) oxidation was done on a chemically de-
fined agar media (CDM) amended with 10 mM sodium arsenite was
used as an electron donor while 5 mM sodium arsenate in the same
medium was used as an electron acceptor, along with 0.5% dextrose,
acting as a carbon source to determine the As (V) reduction ability of
the isolate. The plate was incubated aerobically for 72 h at 30 °C re-
spectively. Flooding the plate with 0.1M silver nitrate (AgNO3) fol-
lowing bacterial growth resulted in the formation of colored precipitate
due to its reaction with As (III) and As (V). A brownish colored pre-
cipitate revealed the presence of silver arsenate (Ag3AsO4) due to the
result of oxidation of As (III) to As (V) (Simeonova et al., 2004). Ar-
senate concentration was measured using ammonium molybdate
method (Dhar et al., 2004).
R. Biswas et al.
2.4. Maximum tolerance concentration (MTC) assay for As and other
heavy metals
The maximum tolerance concentration (MTC) was tested for the
isolate, using the agar dilution method for arsenic as well as for other
heavy metals (Sarkar et al., 2013). Graded concentrations of As salts, As
(III) (NaAsO2) or As (V) (Na2HAsO4.7H2O) were amended to the R2A
agar medium for MTC assay. They were further cultured in R2A media
amended with As [15–70 mM As (III) and 100–1000 mM As (V)].
Moreover, the highly As resistant strain was tested for diverse heavy
metal resistance [ZnCl2, Cu(NO3).23H2O, NiCl2.6H2O, Cd(NO3).24H2O,
HgCl2, and Cr(NO3)3], with varying concentrations of 2.5 mM–30 mM;
[Merck, Germany]. The strain was grown up to log phase and streaked
on each metal amended plate followed by its incubation for 24 h -
36 h at 30 °C.
2.5. Biochemical and physiological characterization
Gram staining was done using the standard Gram staining kit
(Himedia, India). The cellular structure of the isolate was examined
under a bright field microscope. Morphological properties of the se-
lected strain were characterized using Environmental Scanning Electron
Microscopy (ESEM, Quanta FEC 250, Pfeiffer, Netherland) in terms of
its appearance, colony size, surface texture, margin elevation and
general surface structure. Following bacterial growth on R2A media,
temperature and pH sensitivity was monitored at different incubation
temperatures (4 °C - 42 °C) and varying pH (6–10). Standard carbohy-
drate discs (25 μg sugar/disk) and antibiotic discs (Himedia, India)
were used to test the capability of the bacterial strain to metabolize
different carbon sources and to see the diverse antibiotic sensitivity of
the isolate in phenol red agar plates. The oxidative ability of the strain
was tested using standard oxidase discs. Characterization of general
biochemical properties such as indole test, methyl red test, citrate uti-
lization and Voges-Proskauer's test was done using manufacturer's
protocol (Himedia, India). Biofilm formation study was carried out by
spectrophotometric method using dimethyl methylene blue (Pui et al.,
2017).
2.6. Stoichiometry of As (III) oxidation
Arsenite oxidation by the selected strain was observed during
growth of the strain in R2A medium. For studying the growth, the se-
lected strain was grown on R2A medium and incubated at 37 °C for
24 h. The samples were collected in triplicates for every 1 h and optical
density was measured at 600 nm spectroscopically. Standard spectro-
scopic assay of molybdenum blue complex formation was used to de-
termine the concentration of As (V) species in the supernatant (Dhar
et al., 2004). The CFU count for bacterial growth and dry cell biomass
weight were also observed at specific time intervals, followed by stoi-
chiometry and mass balance estimations.
2.7. DNA sequencing and phylogenetic analysis
Isolation of genomic DNA was done using the manufacturer's pro-
tocol (Himedia, India). Amplification of 16S rRNA gene was done using
the genomic DNA of the test strain as a template and 27F (5′-AGAGT
TTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGA-
CTT-3′) as universal primers. PCR was performed with an initial step of
denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation at
the same temperature, annealing at 52 °C for 50 s and elongation at
72 °C for 1.5 min, respectively with a concluding extension step of
10 min at 72 °C. PCR amplified products were purified using Genetix
Gel Extraction Kit (Genetix Biotech Asia Pvt. Ltd., India) followed by
sequencing of 16S rRNA gene from Eurofins sequencing service
(Eurofins Genomics India Pvt Ltd., Bangalore, India). After sequencing,
the sequences were processed using BioEdit tool (BioEdit v7.0.5) and
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International Biodeterioration & Biodegradation 136 (2019) 55–62
blasted in National Center for Biotechnology Information (NCBI) da-
tabase for getting the homology sequences. CLUSTALW multiple
alignments were done to align the retrieved sequences from the NCBI
database along with the sequence of the test strain. Using neighbor-
joining method, the phylogenetic tree was constructed in MEGA-7
(Kumar et al., 2008).
2.8. Arsenate adsorption using natural bio-sorbents
Different bio-sorbents such as red mud, construction site sand,
Moringa oliefera (Drumstick leaves), Psidium guajava (Guava leaves),
Cocos nucifera coir (Coconut coir), Bambusoideae leaves (Bamboo
leaves), sawdust and Sapindus mukorossi (Soap fruit) were used in order
to develop a cost-effective and a natural adsorbent. The samples were
sun-dried and converted to powder form to increase the surface area for
adsorption. A test As (V) solution of 100 mM was prepared and 2 g each
of the collected natural adsorbents were added to the 10 ml of prepared
As (V) stock solution. The As (V) solutions with added adsorbents were
left undisturbed for a period of 15 days. After the incubation period, the
As (V) concentration in the adsorbents was measured using the mo-
lybdenum blue complex formation. The experiment was conducted in
triplicates and the final As (V) concentrations were measured spectro-
scopically. Scanning electron microscopes coupled with energy-dis-
persive X-ray (SEM-EDX) was performed for the two adsorbents,
showing their maximum As (V) adsorption capacities.
2.9. Designing of a two-step approach for total As removal
A two-step experiment was performed in batch condition involving
As (III) oxidizing Delftia spp. BAs29 as oxidizing agent and Moringa
oliefera as bio-sorbent. In the first step, the strain was incubated over-
night at 30 °C in 100 ml of groundwater containing 100 μM As (III) for
evaluating its oxidation ability. In second step, following As (III) oxi-
dation, 2 g of Moringa oliefera was added to the same flask for studying
its adsorption efficiency. Arsenate concentration was finally measured
before and after each step using ammonium molybdate method in the
effluent (Dhar et al., 2004). Bio-adsorption was further confirmed by
SEM-EDX analysis.
2.10. Statistical analysis
All the biological studies have been conducted using triplicate
sampling. The graphs have been represented only after statistical ana-
lysis. Two-way ANOVA analysis in OriginPro 8.5 has been performed
for data representation.
3. Results and discussion
3.1. Isolation and screening of As (III) oxidizing bacteria
Initially, sixty-eight morphologically distinct bacterial strains were
isolated from the As contaminated groundwater samples (Data not
shown). Finally, a highly superior As resistant strain BAs29 was chosen
for further characterization. Bacteria develop some detoxification me-
chanisms to overcome the growth restriction when under selective
stress such as high concentration of As for a prolonged period of time
(Huang et al., 2010). Members of Proteobacteria, such as Hydro-
genophaga and Acidovorax have been identified previously in the con-
taminated groundwater of the region, revealing the high As tolerance
nature of these bacteria (Ghosh et al., 2014).
3.2. Arsenic and other heavy metal tolerance by the selected strain
Silver nitrate screening assay revealed As (III) oxidizing nature of
the strain BAs29. The strain also showed high resistance to As (III) and
As (V) up to 70 mM and 1000 mM. It was further studied for its multi-
R. Biswas et al.
Table 1
Oxidizing efficiency and biomass yield of strain BAs29 with respect to time.
Time(h) Factors
As (III) Oxidizing Bacterial growth Biomass
concentration efficiency (%) (log10 CFU/mL) yield (mg/
(μM) ml)
0 100 0 0.08 0.001
5 97.67 2.33 1.07 0.004
10 71.34 18.66 3.12 0.063
15 19.12 81.37 4.89 0.32
20 3.05 88.67 4.96 0.36
25 1.4 88.72 5.04 0.37
30 1.2 89.14 5.67 0.39
metal tolerance, showing superior resistance up to 30 mM. The strain
showed biofilm forming ability. This phenomenon can be well ex-
plained by the adsorption of heavy metals on the bacterial cell surface,
forming a complex with the exopolysaccharides which can serve as a
major detoxification mechanism (Anwanyu and Ugwul, 2010). With the
increasing concentration of heavy metals, multi-metal resistance ability
of the selected strain will be highly effective in remediating the heavy
metals from contaminated sites. Moreover, the bacteria can gain energy
and enhance their growth rate during the respiratory transformation of
these toxic metals (Oremland and Stolz, 2003). Ubiquitous exopoly-
saccharide from bacterial biomass also serve as an economic, efficient
and sensitive approach of heavy metal ion removal. Having several
controlling factors and distinct dynamics, bio-sorption through exopo-
lysaccharides is a complex phenomenon in itself. Duration of interac-
tion, adsorbent dosage and initial metal ions govern the metal-polymer
interaction. Herminiimonas arsenicoxydans, a gram-negative bacterium
has been found to induce or initiate the formation of biofilm in response
to high As exposure and also scavenge As ions through exopoly-
saccharides (Marchal et al., 2010). Trapped As ions in exopoly-
saccharides induced Thiomonas spp. CB2, a β-proteobacteria; to secrete
biomolecules for the formulation of biofilms (Marchal et al., 2011). It
indicates that in response to heavy metals, these bacteria induce the
synthesis of exopolysaccharides which can effectively be used to adsorb
the heavy metals.
3.3. Physiological and biochemical characterization of the isolate
Physiological analysis revealed that the isolate BAs29 was meso-
philic (30 °C) in nature and survived under slight alkaline environment
(pH-7.5). Previous researches have reported the mesophilic nature
within several As (III) oxidizing bacteria (Kinegam et al., 2008). Strain
BAs29 was seen to be gram-negative and rod-shaped in colony mor-
phology. The strain showed positive result in biofilm formation assay. It
showed negative result to indole and methyl-red test and positive to
Voges-Proskauer's and citrate utilization test. Moreover, it utilized
oxidase to a considerable extent. The antibiotic resistance properties
within the test strain seem to be an interesting finding. Strain BAs29
showed high resistance to Doxycycline hydrochloride, Neomycin, Ce-
fadroxil, Cefotaxime, Ciprofloxacin, Tetracycline, Ceftazidime, Tri-
methoprim, Gentamicin, Chloramphenicol and Cloxacillin while was
sensitive to Vancomycin, Roxithromycin, Ampicillin, Rifampicin, Me-
tronidazole and Norfloxacin. Antibiotic sensitivity of the strain en-
hances its suitable application in bioremediation. Carbohydrate utili-
zation test revealed that the strain BAs29 efficiently utilized multi-
carbons including Sorbitol, Mannitol, Xylose, Maltose, Salicin, and
Fructose. Due to continuous change in physiochemical conditions in
their inherent habitat, the use of various organic carbons is a beneficial
strategy within compelling environments, apart from As (III) coupled
growth for the strains (Sarkar et al., 2014). Under unfavorable condi-
tions, their chances of survival increases by using the limited nutrients
available by acclimatizing their metabolism, making them more
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International Biodeterioration & Biodegradation 136 (2019) 55–62
competing in the microbial community (Hoeft et al., 2007).
3.4. Phylogenetic analysis
The BLAST analysis of partial 16S rRNA gene sequence of the su-
perior As (III) oxidizing strain BAs29 in NCBI showed its homology with
several Delftia spp., particularly with Delftia lacustris, member of β-
proteobacteria belonging to Comamonadaceae family. The 16S rRNA
gene sequence retrieved in this study has been submitted to the NCBI
GenBank with the accession number MH198130. Neighbor-joining
based phylogenetic tree revealed its closeness with several As (III)
oxidizing strains of the same genus including Delftia spp. A2i
(KT835041), Delftia tsuruhatensis T7 (AB075017), Delftia acidovorans
NAT (AM180725), Delftia acidovorans ATCC 15668 (AF078774) and
Delftia TS40 isolated from As contaminated soil, Bangladesh (Cai et al.,
2009; Sultana et al., 2012; Sanyal et al., 2016) (Fig. S1). This genus has
already been previously isolated from diverse environmental sources
such as activated sludge, marine and freshwater, soil, clinical samples
and infected plants (Cai et al., 2009; Sultana et al., 2012; Sanyal et al.,
2016). Arsenic resistant and oxidation ability of this genus is well
known. Furthermore, the phylogenetic tree also revealed its relation
with other heavy metal [Cr (VI), Pb (II), Se (IV) etc.,] resistant, trans-
forming and hydrocarbon degrading Delftia spp. (Prakash et al., 2010;
Juárez-Jiménez, 2010; Ubalde et al., 2012; Wu et al., 2016).
3.5. Arsenite oxidation of the strain BAs29 during growth
Strain BAs29 showed the highest ability of oxidizing in batch test.
The strain showed 91.04% oxidation of 100 μM As (III) during 16 h of
its growth with a doubling time of 1.13 h. Oxidation of As (III) by the
strain was initiated after 6 h of incubation with a considerably long lag
phase (Table 1). The efficiency of As (III) oxidation revealed that more
than 80% of the initial As (III) concentration was oxidized during the
initial log phase [Fig. 2(a)]. Moreover, As (III) supplemented in the
growth medium enhanced the growth up to 40% with respect to control
while the addition of As (V) reduced the growth rate by 12%. Increase
in growth rate during the As (III) oxidation phase, pertains to the fact
that they may gain energy in the presence of As (III), acting as the rate-
limiting step (Fig. S2). This process is indicating the chemolithotrophic
nature of the strain. In this study, the As (III) oxidizing chemolithotroph
gains sufficient energy through the oxidation process of As (III) which is
a thermodynamically exergonic reaction, releasing −67 kJ/reaction of
free energy:
CH2O + 0.704 AsO33−→ 0.235 CH1.79O0.5N0.2 + 0.704 AsO43--
0.126 O2+ 0.79 H2O (1)
where, Biomass yield (YS) = 0.247.
Energetic product yield (EP) = 0.88.
The oxidation of As (III) is done in the periplasm of the bacteria,
catalyzed by As (III) oxidase. The presence of As (III) is sensed by a
sensor kinase AoxS which further activates a regular protein AoxR
(Fig. 3). In association with RpoN (a RNA Polymerase alternative), the
expression of aox operon is controlled by AoxR (Koechler et al., 2010).
There are two basic mechanisms of As (III) extrusion in bacteria. One is
by an As (III) translocating ATPase and the other is via an As (III)
carrier efflux protein where the membrane potential of the cell supplies
the required energy (Oremland and Stolz, 2005). Since the first re-
ported As (III) oxidizing bacterium Bacillus arsenoxydans, several other
As (III) oxidizing strains have been reported till date. In most of the
cases, the isolated bacteria were usually heterotrophs, utilizing As (III)
oxidation as a method of detoxification (Martin and Pederson, 2004).
Mostly the chemolithotrophic bacteria isolated till date belong to the
Agrobacterium or Rhizobium genus (Santini et al., 2000). First time this
R. Biswas et al. International Biodeterioration & Biodegradation 136 (2019) 55–62

Fig. 2. As (III) oxidation potential of the strain BAs29 (a), Lineweaver Burke plot for estimation of kinetic parameters Km and Vmax (b), Luedeking-Piret model for
product formation kinetics (c). Data points are the average of three independent experiments.

59
R. Biswas et al.
Fig. 3. Mechanisms of As (III) oxidation by Delftia spp. BAs29.
study indicates strain BAs29 to be a facultative chemolithotrophic As
(III) oxidizer, utilizing As (III) and glucose as the sole electron donor
and carbon source respectively, validating the experimental calcula-
tions. From the stoichiometric equation, the chemolithotrophic As (III)
oxidation is further confirmed from the negative oxygen demand
coefficient (Eqn. (1)). So, it is clearly vivid that the strain BAs29 utilizes
As (III) instead of oxygen as an electron donor during its chemolitho-
trophic growth. The resultant sigmoid curve with a saturation phase
was obtained when As (III) oxidation rates were plotted as a function of
subsequent As (III) concentrations. The maximum rate of As (III) oxi-
dation (Vmax) by the strain BAs29 was calculated to be 0.657 μM/min,
suggesting an adequate number of As (III) molecules is being catalyzed
per second. A Km value of 21.97 mM is the result of an increasingly
higher affinity towards As (III), indicating that BAs29 gains energy in
the presence of As (III) [Fig. S3 and 2(b)]. Being chemically similar to
phosphate, the toxicity of As (V) oxyanion is primarily based on the
competitive inhibition of proteins that uses phosphate during oxidative
phosphorylation and intermediary metabolism. However, most cells are
comparatively insensitive to As (V) as the intracellular phosphate
concentration is usually quite high within the cells. A modified Monod
model could express the growth of Delftia spp. incorporating inhibitions
by As (V) in the broth culture. The Monod model followed by Lue-
deking-Piret model suggests the mixed-growth associated production of
As (V) during the exponential phase, stating that all the cells in the
juvenile and maturation phase could oxidize As (III) following a uni-
form pattern [Fig. S4 and 2(c)]. Arsenite oxidizing ability was also
observed in Delftia tsuruhatensis T7 (AB075017), Delftia acidovorans
NAT (AM180725) and Delftia acidovorans ATCC 15668 (AF078774)
(Sultana et al., 2012). Klebsiella pneumonia and K. variicola have been
reported to oxidize 87% and 83% of As (III) within 72 h of growth (Butt
and Rehman, 2011). With adherence to the cell membrane or in-
tracellular accumulation, D. tsuruhatensis AR-7 was able to transform Se
(IV) during anaerobic or aerobic growth (Prakash et al., 2010). Delftia
lacustris has previously been reported as a peptidoglycan degrading
bacterium (Jorgensen et al., 2009). A Delftia lacustris strain LZ-C JNCQ
isolated from a petrochemical wastewater discharge was also seen to be
highly resistant to heavy metals (Wu et al., 2016). A Delftia spp. JD2
(EF692532) isolated from soil revealed Cr (VI) and Pb (II) resistance
properties (Ubalde et al., 2012). The ability to degrade or transform
multiple organic pollutants such as diuron, linuron, chloroanilines and
anilines have also been reported earlier by the members of the same
genus (Juárez-Jiménez, 2010).
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International Biodeterioration & Biodegradation 136 (2019) 55–62
3.6. Adsorption of As (V) using natural bio-sorbents
Among all the bio-sorbents used such as Red mud (Riverside),
Construction site sand, Moringa oleifera, Psidium guajava, Cocos nucifera
coir, Bambusoideae leaves, Sawdust and Sapindus mukorossi, Moringa
oleifera showed the maximum As (V) removal capacity of 57.9% from
the As (V) enriched media (100 mM) [Fig. 4(a)]. The highest removal
efficiency was achieved up to 86.25% for 100 mM at pH 6, time 48 h.
Further adsorption isotherm was performed to evaluate the optimum
conditions for As (V) adsorption (Data not shown). The use of ad-
sorbents such as activated carbon, polymer resins, and mineral oxides
have been in commercial use for As bioremediation for a prolonged
period of time but are on the expensive side and their regeneration is
difficult (Sinha et al., 2011). Eguez and Cho reported that activated
charcoal adsorbed 2.5 wt% of As (V), much lower than the natural
adsorbents (Eguez and Cho, 1987). Activated carbon with high ash
content aids the removal of As (V) from contaminated water; being
moderately effective. Presence of other contaminants in the water also
decreases their As (V) adsorption efficiency (Baig et al., 2015). Recent
research showed natural absorbent like rice husk, to be successfully
used for As (V) removal (Asif and Chen, 2017). Therefore, prime im-
portance should be given to affordability, high As removal efficiency,
geographic compatibility, applicability and the reliability of the pro-
cess. These natural adsorbents such as Moringa oleifera, red mud and
guava leaves besides being inexpensive and easily available in many
underdeveloped regions throughout the world, can perform well both
in laboratory and field conditions.
The high As adsorption capabilities by these natural adsorbents can
be attributed to the fact that their phenolic, carbohydrate and the
protein components have functional metal binding groups such as
amino, arsenate sulfate, hydroxyl and carboxyl groups (Mohan and
Pittman, 2007). The complexion of these materials on their surface
pores may enhance the adsorption process or the removal of metal ions
from the solution. Salim et al., used 15 different species of plant for the
removal of Zn from aqueous solutions (Salim et al., 2003). Dried plants
and fruit materials serve as potent low-cost abundant adsorbents as
there is no technicality or environmental stress involved in these ad-
sorbents for As removal.
SEM Analysis revealed the morphological changes within the ad-
sorbent Moringa oleifera [Fig. 4(b) and (c)]. The surface structure of the
adsorbent was uneven and rough before adsorption but post As (V)
adsorption, an enhanced pore structure was revealed, distributed over
the entire surface with a progression of rough activities. The porosity
got increased at a higher temperature due to the disintegration of the
lignocellulosic material and the loss of volatile components. Hence, the
uptake rate of the adsorbent got amplified due to the increase in surface
area by physiochemical activation. Along with As peak, the EDX results
also showed the difference in the amount of oxygen and carbon in the
adsorbent post adsorption [Fig. 4(d) and(e)]. The most effective ad-
sorbent is said to have the least amount of oxygen and the highest
amount of carbon (Xiong et al., 2013). Moringa oleifera revealed more
amount of carbon and As (V) as compared to other tested adsorbents,
implicating Moringa oleifera to be a more effective adsorbent for the
removal of organic pollutants, dyes, and heavy metals from aqueous
solutions. Hence a two-step approach was designed for the removal of
total As content from the groundwater. Initially As (III) oxidizing bac-
teria transformed As (III) into less toxic As (V), followed by its ad-
sorption using cost-effective natural biosorbent Moringa oleifera, aiding
the fabrication of a highly efficient biofilter.
3.7. Two-step approach for As (V) removal
Strain BAs29 showed 96.27% As (III) oxidation potential during
10 h of its growth. Following which, the As (V) containing water was
allowed to adsorb on bio-sorbent Moringa oleifera. The result revealed
presence of only 1.23 μM As (V) concentration in the final test effluent
R. Biswas et al. International Biodeterioration & Biodegradation 136 (2019) 55–62

Fig. 4. Percentage (%) As (V) removal by different adsorbents (a); SEM images of Moringa oleifera before and after As (V) adsorption (b & c); EDX images of Moringa
oleifera before and after As (V) adsorption (d & e).

which indicated an effective As removal efficiency of 98.77% from the Conflicts of interest
groundwater system (Fig. 5).
All the authors of this manuscript declare that there is no conflict of
interest regarding the manuscript submission in the journal of”
4. Conclusion
International Biodeterioration and Biodegradation”.
The current study reveals the first detailed characterization of As
(III) oxidation by Delftia spp. BAs29 in the middle Gangetic plains of
Acknowledgment
Bihar. This strain showed mixed growth associated, facultative che-
molithotrophic nature of As (III) oxidation wherein As (III) and glucose
The authors would like to thank the Department of Science and
were used as the sole electron donor and carbon source respectively, in
Technology, Government of India for providing financial support to the
an exergonic reaction. Besides oxidizing As (III) into less toxic As (V), it
research work (Sanction Order: YSS/2015/001911). We would also like
also showed high multi-metal resistance along with diverse carbon
to express our appreciation to National Institute of Technology,
source utilization, indicating its potential application in various metal
Rourkela for providing conducing laboratory facilities to proceed with
contaminated sites. Maximum As (V) adsorption by Moringa oleifera
the work. The generous help of Prof. Ashok Ghosh during the collection
revealed its suitable utilization in designing of a two-step approach for
of arsenic contaminated water sample from Bhojpur, Bihar is highly
the removal of total As. Hence, Delftia spp. BAs29 along with Moringa
acknowledged.
oleifera will contribute significantly in designing an eco-friendly, cost-
effective in situ bioremediation technology for As contaminated en-
vironments.

61
R. Biswas et al.
Fig. 5. Two-step approach of As (V) removal using Strain BAs29 and Moringa
oleifera.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ibiod.2018.10.006.
References
Aitio, A., Becking, G., 2001. Arsenic and Arsenic Compounds. Environmental Health
Criteria, vol. 224 World Health Organization, Geneva.
Andreoni, V., Zanchi, R., Cavalca, L., Romagnoli, C., Canzi, E., 2012. Arsenite oxidation in
ancylobacter dichloromehanicus As31-1b strain: detection of genes involved in arsenite
oxidation and CO2 fixation. Curr. Microbiol. 65, 212–218.
Anwanyu, C.U., Ugwul, C.E., 2010. Incidence of arsenic resistant bacteria isolated from a
sewage treatment plant. Int. J. Basic Appl. Sci. 10, 64–78.
Asif, Z., Chen, Z., 2017. Removal of arsenic from drinking water using rice husk. Appl.
Water Sci. 7, 1449–1458.
Bahar, M.M., Megharaj, M., Naidu, R., 2012. Arsenic bioremediation potential of a new-
arsenite oxidizing bacterium Stenotrophomonas sp. MM-7 isolated from soil.
Biodegradation 23, 803–812.
Baig, S.A., Sheng, T., Hu, Y., Xu, J., Xu, X., 2015. Arsenic removal from natural water
using low cost granulated adsorbents: a review. Clean. - Soil, Air, Water 43, 13–26.
Banerjee, S., Datta, S., Chattyopadhyay, D., Sarkar, P., 2011. Arsenic accumulating and
transforming bacteria isolated from contaminated soil for potential use in bior-
emediation. J. Environ. Sci. Health Part A 46, 1736–1747.
Butt, A.S., Rehman, A., 2011. Isolation of arsenite-oxidizing bacteria from industrial ef-
fluents and their potential use in wastewater treatment. World J. Microbiol.
Biotechnol. 27, 2435–2441.
Cai, L., Liu, G.H., Rensing, C., Wang, G., 2009. Genes involved in arsenic transformation
and resistance associated with different levels of arsenic-contaminated soils. BMC
Microbiol. 9, 4.
Chakraborti, D., Mukherjee, S.C., Pati, S., Sengupta, M.K., Rahman, M.M., Chowdhury,
U.K., Lodh, D., Chanda, C.R., Chakraborti, A.K., Basu, G.K., 2003. Arsenic ground-
water contamination in Middle Ganga Plain, Bihar, India: a future danger? Environ.
Health Perspect. 111, 1194–1201.
Dai, X., Ping, L., Jin, T., Rui, Z., Dazhuni, W., Bing, L., Yanhong, W., Zhou, J., 2016.
Evidence of arsenic mobilization mediated by an indigenous iron reducing bacterium
from high arsenic groundwater aquifer in Hetao Basin of Inner Mongolia, China. Int.
Biodeterior. Biodegrad. 128, 22–27.
Dhar, R.K., Zheing, Y., Rubenstone, J., Geen, A.V., 2004. A rapid colorimetric method for
measuring arsenic concentrations in groundwater. Anal. Chim. Acta 526, 203–209.
Eguez, H.E., Cho, E.H., 1987. Adsorption of arsenic on activated charcoal. JOM (J. Occup.
Med.) 39, 38–41.
Ghosh, D., Bhadury, P., Routh, J., 2014. Diversity of arsenite oxidizing bacterial com-
munities in arsenic-rich deltaic aquifers in West Bengal, India. Front. Microbiol. 5,
602.
Gu, J.D., 2018. Mining, pollution and site remediation. Int. Biodeterior. Biodegrad.
128, 1–2.
Gu, J.D., Wang, Y., 2013. A new era for geomicrobial ecotoxicology in environmental
science research. Int. Biodeterior. Biodegrad. 76, 1–2.
Hoeft, S.E., Switzer Blum, J.I., Stolz, J.F., Tabita, F.R., Witte, B., King, G.M., Santini, J.M.,
Oremland, R.S., 2007. Alkalilimnicola ehrlichii sp. nov., a novel, arsenite-oxidizing
haloalkaliphilic gammaproteobacterium capable of hemoautotrophic or hetero-
trophic growth with nitrate or oxygen as the electron acceptor. Int. J. Syst. Evol.
Microbiol. 57, 504–512.
62
International Biodeterioration & Biodegradation 136 (2019) 55–62
Huang, A., Teplitski, M., Rathinasabapathi, B., Ma, L., 2010. Characterization of arsenic-
resistant bacteria from the rhizosphere of arsenic hyperaccumulator Pterisvittata. Can.
J. Microbiol. 56, 236–246.
Jorgensen, N.O., Brandt, K.K., Nybroe, O., Hansen, M., 2009. Delftia lacustris sp. nov., a
peptidoglycan-degrading bacterium from fresh water, and emended description of
Delftia tsuruhatensis as a peptidoglycan-degrading bacterium. Int. J. Syst. Evol.
Microbiol. 59, 2195–2199.
Juárez-Jiménez, B., Manzanera, M., Rodelas, B., Martínez-Toledo, M.V., Gonzalez-López,
J., Crognale, S., Pesciaroli, C., Fenice, M., 2010. Metabolic characterization of a strain
(BM90) of Delftia tsuruhatensis showing highly diversified capacity to degrade low
molecular weight phenols. Biodegradation 21, 475–489.
Kamaluddin, S.P., Arunkumar, S., Ramasamy, K., 2003. Bioremediation of chromium
contaminated environments. Indian J. Exp. Biol. 41, 972–985.
Kinegam, S., Yingprasertchai, T., Tanasupawat, S., Leepipatpiboon, N., Akaracharanya,
A., Kim, K.W., 2008. Isolation and characterization of arsenite-oxidizing bacteria
from arsenic-contaminated soils in Thailand. World J. Microbiol. Biotechnol. 24,
3091–3096.
Koechler, S., Cleiss-Arnold, J., Proux, C., Sismeiro, O., Dillies, M.A., Goulhen-Chollet, F.,
Hommais, F., Lièvremont, D., Arsène-Ploetze, F., Coppée, J.Y., Bertin, P.N., 2010.
Multiple controls affect arsenite oxidase gene expression in Herminiimonas arseni-
coxydans. BMC Microbiol. 10, 53.
Kumar, S., Dudley, J., Nei, M., Tamura, K., 2008. MEGA: a biologist-centric software for
evolutionary analysis of DNA and protein sequences. Briefings Bioinf. 9, 299–306.
Lim, K.T., Shukor, M.Y., Wasoh, H., 2014. Physical, chemical, and biological methods for
the removal of arsenic compounds. BioMed Res. Int. 9–16.
Liu, F., Guoping, Z., Shirong, L., Zhiping, F., Jingjing, C., Chao, M., 2018. Bioremoval of
arsenic and antimony from wastewater by a mixed culture of sulfate-reducing bac-
teria using lactate and ethanol as carbon sources. Int. Biodeterior. Biodegrad. 126,
152–159.
Marchal, M., Briandet, R., Koechler, S., Kammerer, B., Bertin, P.N., 2010. Effect of ar-
senite on swimming motility delays surface colonization in Herminiimona sarseni-
coxydans. Microbiology 156, 2336–2342.
Marchal, M., Briandet, R., Halter, D., Koechler, S., DuBow, M.S., Lett, M.C., Bertin, P.N.,
2011. Subinhibitory arsenite concentrations lead to population dispersal in
Thiomonas sp. PLoS One 6, 23181.
Martin, A.J., Pedersen, R.F., 2004. Alteration to lake trophic status as a means to control
arsenic mobility in a mine-impacted lake. Water Res. 381, 4415–4423.
Mohan, D., Pittman, C.U.J., 2007. Arsenic removal from water/wastewater using ad-
sorbents – a critical review. J. Hazard Mater. 142, 1–53.
Oremland, R.S., Stolz, J.F., 2003. The ecology of arsenic. Science 300, 939–944.
Oremland, R.S., Stolz, J.F., 2005. Arsenic, microbes and contaminated aquifers. Trends
Microbiol. 13, 45–49.
Paul, D., Kazy, S.K., Banerjee, T.D., Gupta, A.K., Pal, T., Sar, P., 2015. Arsenic bio-
transformation and release by bacteria indigenous to arsenic contaminated ground-
water. Bioresour. Technol. 188, 14–23.
Prakash, D., Pandey, J., Tiwary, B.N., Jain, R., 2010. Physiological adaptations and tol-
erance towards higher concentration of selenite (Se+4) in Enterobacter sp. AR-4,
Bacillus sp. AR-6 and Delftia tsuruhatensis Ar-7. Extremophiles 14, 261–272.
Pui, C.F., Apun, K., Jalan, J., Bilung, L.M., Suut, L., Hashim, H.F., 2017. Microtitre plate
Assay for the quantification of biofilm formation by pathogenic leptospira.
Microbiology 12, 146–153.
Salim, R., Al-Subu, M., Abu-Shqair, I., Braik, H., 2003. Removal of zinc from aqueous
solutions by dry plant leaves. Process Saf. Environ. Protect. 81, 236–242.
Santini, J.M., Sly, L.I., Schnagl, R.D., Macy, J.M., 2000. A new chemolithoautotrophic
arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological,
and preliminary biochemical studies. Appl. Environ. Microbiol. 66, 92–97.
Sanyal, S.K., Mou, T.J., Chakrabarty, R.P., Hoque, S., Hossain, M.A., Sultana, M., 2016.
Diversity of arsenite oxidase gene and arsenotrophic bacteria in arsenic affected
Bangladesh soils. Amb. Express 6, 21.
Sarkar, A., Kazy, S.K., Sar, P., 2013. Characterization of arsenic resistant bacteria from
arsenic rich groundwater of West Bengal, India. Ecotoxicology 22, 363–376.
Sarkar, A., Kazy, S.K., Sar, P., 2014. Studies on arsenic transforming groundwater bacteria
and their role in arsenic release from subsurface sediment. Environ. Sci. Pollut. Res.
Int. 14, 8645–8662.
Simeonova, D.D., Lievremont, D., Lagarde, F., Muller, D.A.E., Groudeva, V.I., Lett, M.C.,
2004. Microplate screening assay for the detection of arsenite-oxidizing and arsenate-
reducing bacteria. FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Lett. 237, 249–253.
Sinha, S., Amy, G., Yoon, Y., Her, N., 2011. Arsenic removal from water using various
adsorbents: magnetic ion exchange resins, hydrous ion oxide particles, granular ferric
hydroxide, activated alumina, sulfur modified iron, and iron oxide-coated microsand.
Environ. Eng. Res. 16, 165–173.
Sultana, M., Vogler, S., Zargar, K., Schmidt, A.C., Saltikov, C., Seifert, J., Schlömann, M.,
2012. New clusters of arsenite oxidase and unusual bacterial groups in enrichments
from arsenic-contaminated soil. Archiev. Microbiol. 194, 623–635.
Tripti, K., Shardendu, 2017. Arsenic removing soil indigenous bacteria of hyper arsenic
contaminated region in Bihar. Proc. Natl. Acad. Sci. India B Biol. Sci. 1–9.
Ubalde, M.C., Braña, V., Sueiro, F., Morel, M.A., Martínez-Rosales, C., Marquez, C.,
Castro-Sowinski, S., 2012. The versatility of Delftia sp. isolates as tools for bior-
emediation and biofertilization technologies. Curr. Microbiol. 64, 597–603.
Wu, W., Huang, H., Ling, Z., Yu, Z., Jiang, Y., Liu, P., Li, X., 2016. Genome sequencing
reveals mechanisms for heavy metal resistance and polycyclic aromatic hydrocarbon
degradation in Delftia lacustris strain LZ-C. Ecotoxicology 25, 234–247.
Xiong, C., Jia, Q., Chen, X., Wang, G., Yao, C., 2013. Optimization of polyacrylonitrile-2-
aminothiazole resin synthesis, characterization, and its adsorption performance and
mechanism for removal of Hg (II) from aqueous solutions. Ind. Eng. Chem. Res. 52,
4978–4986.