Term paper

On

ARSENIC REMOVAL FROM GROUND WATER USING

MICROBIOLOGY

Submitted by

Sanjay Kumar

20062040

Environmental Engineering

Under The Guidance

of

Dr. Devendra Mohan

Professor and Former Head
Department of Civil Engineering
Indian Institute of Technology (Banaras Hindu University)
Varanasi –221005.

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 ACKNOWLEDGEMENT
I am highly indebted to my teacher Professor Devendra Mohan for his guidance and persistent
directions as well as for providing me the chance to work on this topic & also for the support in
completing this work.

I would like to express my courtesy and heartily thanks to my parents for their belief in me and
for the encouragement which kept me motivated to complete this work.

I would also like to vote my thanks to all my teachers of Environmental Engineering,

Department of Civil Engineering, Indian Institute of Technology BHU for their valuable
comments and suggestions.

My thanks and appreciations also go to my seniors and my fellow batch mates in completing
this work and people who have assisted me throughout with their knowledge and abilities.

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 CONTENTS

Abstract----------------------------------------------------------------------------------------- 4

Introduction ----------------------------------------------------------------------------------- 4

Sources of arsenic contamination----------------------------------------------------- 6

Figure 1: Major Sources of Arsenic ------------------------------------------------------------------ 7

BIOREMEDIATION------------------------------------------------------------- 7

Direct microbial bioremediation-------------------------------------------------- 8

Indirect microbial bioremediation---------------------------------------------- 8

Arsinite Oxidation-------------------------------------------------------------- 9

Arsenate Reduction------------------------------------------------------------- 11

Arsenic removal from water: Biological step ---------------------------- 12

Conclusion and Future Perspective----------------------------------------- 14

Reference ------------------------------------------------------------------------- 16

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 ABSTRACT

Groundwater plays a central role in the hydrological cycle and represents the utmost natural
resource for human consumption and activities on a global scale. Therefore, any source of
contamination of either geogenic or anthropogenic origin may provide a serious environmental
health threat. Within the long list of organic and inorganic groundwater contaminants, arsenic, a
toxic element retrieved in air, soils, rocks, waters and organisms, can occur at high
concentrations in aquifers representing an issue of worldwide concern. High arsenic
concentrations in groundwater in different states of India have become a major cause of concern
in recent years. The groundwater in the past was considered to be safe for drinking purposes, but
now it is recognized that many shallow tube wells contain arsenic at concentrations higher than
the safe limit set for drinking purpose by WHO (1993). It is becoming an emerging issue in the
water supply and health related sectors of India. The purpose of this paper is to review the recent
status of arsenic in groundwater in Chunar Mirzapur district and present suitable methods to be
adopted in mitigating its adverse effects on human health. Over the past years, research efforts
aimed to elucidate the microorganisms and mechanisms involved in the biogeochemical cycling
of this element. This review highlights the current understanding of the ecology, biochemistry
and genomics of the various microorganisms, and their potential application in the treatment of
arsenic-polluted water. Moreover, a broad evaluation of the application potentialities of
microbiological processes suitable for treatment strategies of arsenic contaminated groundwater
is provided.

INTRODUCTION

Drinking water access is one of the most fundamental of all human rights; however, many millions
of people around the world, particularly in low-income, developing countries, are regularly exposed
to water sources that are contaminated by pathogenic bacteria, or toxic levels of pollutants such as
the arsenic (As). Arsenic (As) is a metalloid element (atomic number 33) with one naturally
occurring isotope of atomic mass 75, and four oxidation states (-3, 0, +3, and +5). In the aqueous
environment, the +3 and +5 oxidation states are most prevalent, as the oxyanions arsenite
(H3AsO3 or H2AsO3– at pH ~9-11) and arsenate (H2AsO4– and HAsO42- at pH ~4-10) (Smedley
and Kinniburgh, 2002). Arsenic is widely distributed in nature and principally occurs in the form
of inorganic or organic compounds. In soils, arsine gases may be generated by fungi and other
related micro-organisms. Different forms of arsenic have different toxicities, with arsine gas
being the most toxic form of the inorganic oxyanions, arsenite are the most toxic forms and
arsenate, the less toxic forms. The organic (methylated) arsenic forms are considered least toxic.
Exposure to inorganic compounds may occur in a number of ways through certain industrial
effluents, pesticides, chemical alloys, combustion of fossil fuels, wood preservative agents,
occupational hazards in mining and dissolution in drinking water. The most commonly found

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arsenic compounds in drinking water are arsenate or arsenite. However, groundwater is
notoriously prone to chemical and other types of contamination from natural sources or by
anthropogenic activities.
Reliable information on arsenic vulnerability and health effects are seldom available, but it is
clear that in many nations of the world, arsenic in drinking water has been observed at
concentrations greater than the WHO guideline value i.e. 10 µg/l (0.01 mg/l) or the prevailing
national standards. The major issue of arsenic contaminated water is to find out the level of
contamination and it is not so easy because of no colour, no odour and no taste even in the highly
contaminated water. Arsenite is more toxic (approximately ten times) than the arsenate due to the
former’s ability to react with sulfhydryl groups thereby increasing the residence time (Nagarnaik
et al., 2002).

A sustainable approach to addressing these contamination issues is to either change the water
source or treat the water source before it is consumed. Often, however, simply changing the
water supply is not an option as entire regions may be affected and it becomes economically
unviable to transport clean water over many hundreds of kilometres. Instead, it may be easiest
and cheapest to treat the water before it is drunk or used for other purposes (e.g. agriculture,
washing, etc.). Many technologies are now available for arsenic removal that have been
specifically developed for industrial-scale plants; among these, the best available technologies
include anion exchange, activated alumina, reverse osmosis, modified coagulation/filtration,
modified lime softening and oxidation/filtration. Among the removal processes with the highest
efficiency (>95% of arsenic removal), those using sorbents such as activated alumina turned out
to be the cheapest. Other commonly used sorbents with high arsenic removal efficiency (>95%)
are based on iron compounds. These sorbents are not regenerable and their adoption remains
expensive for poor countries. For this reason, research for cost-effective and efficient alternative
sorbents (or surface-coated sorbents) is still in progress. Moreover, any effective treatment of
arsenic contaminated water has to remove both As(III) and As(V) forms, but sometimes classical
technologies are not efficient enough in the removal of As(III), as As(III) is more difficult to
remove by the positively charged surfaces of adsorbents. This is a crucial point because the new
arsenic drinking water standard imposes a reduction of arsenic to 10 μg/l and, consequently,
there is a dramatic demand for oxidation technologies that effectively convert As(III) into As(V)
before its removal.

Currently, the arsenic contamination in groundwater has been widely studied from the
geochemical point of view, but less is known about the microbiology of arsenic in such
environments. Studies performed so far highlighted the presence of a variety of arsenic-resistant
and transforming bacterial species in groundwater (Corsini et al., 2014; Davolos and Pietrangeli,
2011; Li et al., 2016; Liao et al., 2011; Sarkar et al., 2013; Wang and Mulligan, 2006). However,
as detailed in the following, the biodiversity and metabolic potentialities of the microbial
communities of such arsenic-rich aquatic environments are still poorly understood. Biological
water treatment methods are considered to be a suitable approach to overcome above mentioned

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problems and they have attracted considerable research interest over recent years. Removal of
arsenic can be performed by using natural consortia, pure cultures of arsenic resistant bacteria or
iron- and manganese-oxidizing bacteria that can transform and/or capture arsenic forms
indirectly.
Microorganisms can mediate redox transformations of As via As(V) reduction and As(III)
oxidation (Oremland and Stolz, 2003). To date, a wide variety of As(V)-reducing and As(III)-
oxidizing prokaryotes have been isolated from various As-contaminated environments
(Oremland and Stolz, 2005, 2003), and a recent study suggested that they are also distributed in
the natural environment containing background levels of As. Because both As(V) reduction and
As(III) oxidation directly affect the mobility and bioavailability of As, microbial activities play a
key role in biogeochemical As cycling. Such microbial processes have the potential to promote
As removal from contaminated soils/waters when used as intended.

This article outlines the latest physiology and phylogeny for microbial metabolism of inorganic
As and highlights their advances as bioremediation techniques which make use of
microbiological processes in order to remove As from drinking water.

Sources of arsenic contamination

Arsenic is a natural constituent of the earth’s crust and is the 20th most abundant element. The
average concentration of arsenic in the continental crust is 1–2 mg/kg（Taylor and McLennan,
1985). Arsenic is released into the environment through natural processes such as weathering and
volcanic eruptions, and may be transported over long distances as suspended particulates and
aerosols through water or air. A range of arsenic compounds, both organic and inorganic, are
introduced into the environment through geological (geogenic) and anthropogenic (human
activities) sources (Figure 1). Small amounts of arsenic also enter the soil and water through
various biological sources (biogenic) that are rich in arsenic. Although the anthropogenic source of
arsenic contamination is increasingly becoming important, it should be pointed out that the recent
episode of extensive arsenic contamination of groundwater in Bangladesh and West Bengal is of
geological origin, transported by rivers from sedimentary rocks in the Himalayas over tens of
thousands of years, rather than anthropogenic.

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Figure 1: Major Sources of Arsenic

source:http://advancejournals.org/The-International-Journal-of-Earth-and-Environmental-
Sciences/article/status-and-mitigation-of-arsenic-contamination-in-groundwater-in-india/

BIOREMEDIATION

Bioremediation strategies where bacteria are actively involved with the transformation of a
species (e.g. through oxidation or reduction) are referred to it herewith as ‘direct microbial
bioremediation’, whereas treatments by secondary processes (e.g. mineralization) stimulated by
bacteria are referred to as ‘indirect microbial bioremediation’.

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Direct microbial bioremediation

Direct bioremediation approaches for As include those which directly reduce As(V) or oxidize
As(III), which can be subsequently sorbed to or sequestered in minerals. As(III) oxidation can be
mediated by heterotrophic bacteria, which use a detoxification mechanism to convert As(III) to
As(V). Such strains include Alcaligenes sp. (Osborne et al., 1976). and Variovorax sp. MM-1,
which was noted to show the highest oxidation rates of all heterotrophs (1.23 x 10-7 μM-1 min-1
cell-1) (Bahar et al., 2016). Autotrophic As(III)-oxidizing bacteria use As(III) as an electron
donor, coupled with O2 as an electron acceptor and CO2 as a carbon source. As(V) reduction has
also been seen to take place as a detoxification strategy of bacteria via cytoplasmic arsenate-
reductase enzymes, although As(V) can also be used as a terminal electron acceptor for
respiration, leading to As(III) remobilization, which is then removed by precipitation or
complexation with sulfides (Bahar et al., 2016). Such a sequestration strategy, however, would
only be suitable in environments where sulfide and arsenic are present.

Indirect microbial bioremediation

Indirect microbial bioremediation includes pathways, which do not enzymatically modify the As
species, but instead stimulate other processes (e.g. iron oxidation) in which minerals are formed
that subsequently sorb or sequester the heavy metals. Such mechanisms are evident in North
Vietnam, where local people have developed household filters which simply contain sand. Iron-
and arsenic-containing contaminated groundwater is added at the top and then collected after
filtration with an average of 80% As removed. The filters have relatively humble origins and
were initially introduced to curb the unpleasant taste of the iron, which was characteristic of the
local water supply (Berg et al., 2007). A rather serendipitous side-effect was that as iron
precipitated through abiotic and biotic iron oxidation, arsenic species bound to and were
incorporated within the iron minerals (Berg et al., 2007; Voegelin and Hug, 2003). This effect
was not only rapid but highly effective with a common household drinking water filterable to
supply enough water for a small household in just a few minutes. Disadvantages of these filters
include the fact that there are insufficient disposal methods available for the used filter material,
meaning that As-loaded iron rich sand is usually not properly disposed of and is simply
discarded in the garden, street or river. Also, there is a risk that the presence of organic matter in
the filter material could stimulate Fe(III)-reducing bacteria, which can subsequently promote
dissolution of the Fe minerals and release of As, especially after disposal of the used filter
material (Islam et al., 2005). Another disadvantage of such filters includes the fact that they are
only suitable when the ratio of Fe is high enough (Fe/As is ≥ 50) in the groundwater to form
sufficient sorption site on fresh Fe mineral precipitates (Berg et al., 2007). Furthermore, whilst
such an approach appears suitable for small-scale applications, the ability to purify large volumes
of water which could subsequently be used for agriculture remains out of reach. Alternative

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strategies for heavy metal bioremediation consider the possible incorporation of As into different
iron minerals such as magnetite. Magnetite is a magnetic, mixed-valent iron mineral (containing
both Fe2+ and Fe3+) which has been shown to incorporate both As(V) and As(III) produced by
microbial Fe(III) reduction by Geobacter sulfurreducens (Islam et al., 2005). The magnetic
nature of the mineral means that it can be used to treat the contaminated water supply and either
be removed by magnetic separation, or kept in place using a magnetic trap. Furthermore, the
ability to control the particle size of biogenic magnetite particles (Nitzsche et al., 2015) or
selectively promote the formation of other biogenic iron minerals such as goethite (Kleinert et
al., 2011) means that biogenic approaches could be used to tailor the reactivity of biogenic iron
minerals to selected bioremediation applications. The problem associated with the
aforementioned studies, however, is that they were predominantly done ex-situ and can thus be
only done following extraction of the water from the aquifer. Sustainable strategies should
perhaps instead focus on trying to promote the safe remediation of drinking water supplies in
situ.

Arsenite oxidation

Microbial As(III) oxidation has been described as either a detoxification mechanism (Christoph
Mueller et al., 2003) or an energy source for chemolithoautotrophic microbes (Garcia-
Dominguez et al., 2008; Shelley E. Hoeft et al., 2007). The process is performed by various
groups of bacteria and archaea which include both heterotrophic As(III) oxidizers and
chemolithoautotrophic As(III) oxidizers. The heterotrophic oxidation of As(III) (Eq. 1) is
considered primarily as a detoxification reaction that converts As(III) encountered on the cell’s
outer membrane into the less toxic form, As(V), without obtaining energy from the exergonic
reaction (Santini et al., 2002).

2− +
2H3AsO3 + O2 HAsO4 + H2As𝑂4−+ 3H (ΔG0 = -256 KJ/Rx)

So far, only a small number of bacteria (i.e., Pseudomonas arsenitoxidans; strain NT-26) have
been described for the capability to use the energy gained from this reaction for cell growth
(Eq. 2)
− +
H3AsO3 + ½ O2 H2AsO4 + H (ΔG0 = -113.34 KJ/Rx)

These microorganisms were found to grow chemolithoautotrophically with arsenite as electron

donor, oxygen as an electron acceptor and CO2 as the carbon source for fixation (Santini et al.,
2002).

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Within heterotrophic As(III) oxidizers, some facultative anaerobic bacteria capable of either
aerobic As(III) oxidation or anaerobic As(V) reduction were reported (Gihring and Banfield,
2001; Handley et al., 2009). Bacteria can oxidize As(III) to As(V) through the enzyme arsenite-
oxidase, which was first isolated in 1992. This enzyme, first purified and characterized from
Alcaligenes faecalis (Naujokas et al., 2013), is composed of a small subunit containing a Rieske
[2Fe-2S] cluster and a large subunit harbouring molybdopterin guanosine dinucleotide at the
active site and a [3Fe-4S] cluster (Ellis et al., 2006). The enzyme is involved either in arsenic
detoxification by heterotrophic bacteria (Christoph Mueller et al., 2003) or in energy generation
by chemolithoautotrophic bacteria (Oremland and Stolz, 2005; Santini et al., 2002). The genes
encoding these two subunits were first identified and sequenced in the heterotrophic bacterium
H. arsenicoxydans strain ULPAs1 (Christoph Mueller et al., 2003).

Although homologous genes encoding these two subunits were formerly assigned different
names (aoxB-aoxA/aroA-aroB/asoA-asoB), nomenclature for genes involved in prokaryotic
aerobic As(III) oxidation was recently unified and the name aio was newly assigned; therefore,
the large and small subunit are denoted aioA and aioB, respectively (Lett et al., 2012). AioA is
similar to the molybdenum-containing subunits in the DSMO reductase family and distantly
related to the catalytic subunit of respiratory As(V) reductase (ArrA) (Islam et al., 2005; Silver
and Phung, 2005a; Stolz et al., 2010). Homologs of genes encoding AioA are found in
phylogenetically diverse strains including members of a-, b-, g-Proteobacteria, Bacteroidetes,
Actinobacteria, Firmicutes, Aquificae, Deinococcus-Thermus, Chlorobi, Chloroflexi, Nitrospira,
and Crenarchaeota. These genes are found clustered in several groups in the AioAbased tree,
with strains in the phyla including thermophiles forming distinct phylogenetic branches from
mesophiles. The major mesophile branches are divided into two groups, group I, which is
mainly composed of a-Proteobacteria, and group II, which is primarily composed of b- and g-
Proteobacteria. This pattern suggests that these groups probably originated from respective
proteobacterial divisions. However, there are considerable inconsistencies between the AioA-
based phylogeny and 16S rRNA based classification, suggesting that horizontal gene transfer
plays a role in the propagation of aio genes in prokaryotes (Heinrich-Salmeron et al., 2011).
aioA-like genes have been amplified from a variety of As-rich environments including mine,
arsenical pesticide- or smelter impacted sites, and geothermal sites (Cai et al., 2009; Hamamura
et al., 2008; Inskeep et al., 2007). These investigations suggest that the diversity of aioA genes
in prokaryotes is wider than previously suspected. Moreover, aioA-like genes have been
obtained from soil or sediments containing background levels of As, indicating that diverse
aerobic As(III) oxidizers reside in the environment, regardless of As contamination.

An anaerobic As(III)-oxidizing bacterium was isolated (Oremland et al., 2002), strain MLHE-1,
from anoxic bottom water of Mono Lake, CA, USA, which is an alkaline soda lake known for its
high concentration of As(V) (200 mM). Strain MLHE-1, later proposed as Alkalilimnicola

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ehrlichii sp. nov.(Shelley E Hoeft et al., 2007) , is a chemolithoautotrophic bacterium that can
couple As(III) oxidation to nitrate reduction under anaerobic conditions. A purple sulfur
bacterium, Ectothiorhodospira sp. PHS- 1, was also isolated from red-pigmented biofilms in
Mono Lake (Schoepp-Cothenet et al., 2009). This strain can use As(III) as the electron donor for
anoxygenic photosynthesis and produces As(V) anaerobically under light conditions.
Interestingly, both of these bacteria appear to lack aioA genes and instead possess genes that are
much more closely related to arrA (Stolz, 2016; Zargar et al., 2010). This gene, designated arxA,
is required for chemoautotrophic growth on As(III) and nitrate by strain MLHE-1. In addition,
arxA is strongly induced by As(III) in strain PHS-1. Thus, it is possible that ArxA is a novel type
of As(III) oxidase that forms a distinct phylogenetic clade within the dimethyl sulfoxide
(DMSO) reductase family. arxA-like genes have recently been found in a nearly complete
genome sequence of the uncultured bacterium within the candidate division OP1 (Takami et al.,
2012) and in a reconstructed complete genome of the dominant organism (RBG-1) in deep
sediment of the Colorado River, CO, USA (Castelle et al., 2013). As(III)-oxidizing denitrifying
chemoautotrophs (strains DAO-1 and DAO-10 within the classes b- and a-Proteobacteria,
respectively) have been isolated from As-contaminated soils, but it is still unclear which genes
are required for As(III) oxidation.

Arsenate reduction

As(V) reduction as a detoxification system Arsenic resistant microorganisms are able to cope
with high arsenic concentrations (Nriagu et al., 2007). As(V) is reduced to As(III) within the cell
membrane by a cytoplasmic As(V) reductase (arsC) through a detoxification process (Páez-
Espino et al., 2009) found widely in microbes. Arsenic resistance in prokaryotes is conferred by
the arsRBC operon (Silver and Phung, 2005b) whereas arsR and arsC genes encode for the
regulator and cytoplasmic As(V) reductase respectively. After As(V) reduction, (Kruger et al.,
2013)As(III) is finally excreted out of the cells via a membrane efflux pump encoded by arsB
gene (Rosen, 2002). This gene operates as a uniporter using the membrane potential to extrude
arsenite and antimonite oxyanions (Meng et al., 2004). Supplementary genes may also be
associated with ars operon; arsA codes for an ATPase which binds to arsB and makes the As(III)
efflux more efficient whereas arsD encodes for a protein which acts as an As(III) chaperone
(Kruger et al., 2013). This enlarged arsRDABC operon has been found in fewer bacterial
genomes than arsRBC ones. The presence of at least one of arsH, arsN, arsP, arsTX, arsO and
glo genes was recorded in some microorganisms (e.g., Campylobacter jejuni, Microbacterium
spp.). These genes seem to be involved in arsenic resistance with accessory functions such as the
encoding for a putative membrane permease, a thioredoxin system and an acetyltransferase-like
protein (Li et al., 2014). To date, the role of these genes in arsenic resistance has been not yet
completely elucidated, since other unknown resistance processes might be involved (Kruger et
al., 2013). Less is known about the second family of As(III) carriers, acr3pA (also called arr3p),

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that contains acr3 gene which has the same function of arsB, a component of the more common
detoxifying operon (Achour et al., 2007). Although members of the family acr3pA were found in
bacteria, archaea and fungi (Rosen, 2002; Wysocki et al., 2003), they were functionally
characterized only in few species such as Bacillus subtilis, Synechocystis sp., Corynebacterium
glutamicum and Saccharomyces cerevisiae (Sato and Kobayashi, 1998; Wysocki et al., 1997). It
is worth noting that arsB is restricted to prokaryotes, whereas acr3 homologs are present in
eukaryotes (Rosen, 2002).

In addition to the detoxifying As(V) reduction, certain bacteria can reduce As(V) as the terminal
electron acceptor for anaerobic respiration. Such bacteria are defined as dissimilatory As(V)-
reducing prokaryotes (DARPs). These bacteria are phylogenetically diverse, including members
of Firmicutes, g-, d-, and ε-Proteobacteria (Oremland and Stolz, 2005).

As sorption onto metal oxide minerals, especially on iron (hydr) oxides, is an important process
controlling the dissolved concentration of As in various environments. As(V) is strongly
associated with iron and aluminum (hydr)oxides, whereas As(III) is more mobile than As(V).
Thus, reductive dissolution of As-bearing iron (hydr)oxides by dissimilatory iron-reducing
bacteria can cause As release (Dixit and Hering, 2003). In addition, direct reduction of As(V)
adsorbed onto soil minerals by DARPs may be another important mechanism of As mobilization
(Cummings et al., 1999).

Arsenic removal from waters: the biological step

Innovative technology for arsenic removal uses bio column reactors consisting of immobilized
bacterial cells capable of arsenic adsorption. A novel, cost-effective biocomposite – granules of
cement coated with cysts of Azotobacter – has been used for arsenic removal from drinking
water (Gauri et al., 2011). This biocomposite removed approximately 96% of arsenic, probably
due to the presence of polysaccharides and other macromolecules that interact with arsenic. Cells
of Ralstonia eutropha immobilized on a granular, activated carbon bed in a column reactor to
remove arsenic from a synthetic industrial effluent (Mondal et al., 2008). After an initial stage of
adaptation and biofilm formation, the cells were able to capture both As(III) and As(V).

Bioremoval processes involve both the direct adsorption of arsenic by microbial biomass and the
adsorption and coprecipitation of arsenic with biogenic iron or manganese hydroxides (Wang
and Zhao, 2009). The application of biological processes for the oxidation and removal of
dissolved iron and manganese has been proposed as another efficient means for the simultaneous
removal of arsenic and iron. The main product of biological oxidation of iron is usually a
mixture of poorly ordered iron oxides with significant amounts of organic matter. Arsenic can be
removed by direct adsorption or by coprecipitation on the preformed biogenic iron oxides,
whereas there is also an indication of As(III) oxidation by iron-oxidizing bacteria, leading to
improved overall removal efficiency. Investigation regarding the removal of arsenic during
biological iron oxidation in a fixed-bed upflow filtration unit containing polystyrene beads was

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also done (Katsoyiannis et al., 2004). Iron oxides were deposited in the filter medium, along with
the iron-oxidizing bacteria Gallionella ferruginea and Leptothrix ochracea, offering a favourable
environment for arsenic to be adsorbed and consequently removed from the aqueous streams.
The authors also demonstrated that, under the experimental conditions used, As(III) was oxidized
by microorganisms that colonized the filter medium, contributing to an overall increase of
arsenic removal (up to 95%), even when initial arsenic concentrations were up to 200 μg/l.

As(V) reducers were thought to increase the element’s mobility until the discovery of
Desulfosporosinus auripigmenti, an As(V)- and sulfur-respiring microorganism that precipitates
arsenic trisulfide (As2S3), leading to the biogeogenic formation of auripigment (Katsoyiannis et
al., 2004). More recently, photoactive As–S (‘realgar’) nanotubes have been shown to be
produced by Shewanella sp. strain HN-41, an anaerobic bacterium that uses S2𝑂32− as an
electron acceptor and lactate as an electron donor, and concomitantly reduces As(V) to As(III)
for detoxification purposes (Newman et al., 1997).

Besides bioremoval of arsenic and biogeogenic mineral formation, bacterial oxidation of As(III)
to As(V) is a promising approach to treat contaminated water instead of using conventional
oxidants (i.e., potassium permanganate, chlorine, ozone, hydrogen peroxide or manganese
oxides).

In recent years, several studies have been conducted to assess the As(III) oxidation efficiency of
different As(III)-oxidizing bacteria attached on immobilized materials. A bioreactor with Ensifer
adhaerens cells was developed which was immobilized on polyvinyl alcohol gel droplets to
study the As(III) oxidation efficiency of the strain in synthetic groundwater containing 1 mg/l of
As(III) (Lee et al., 2007). The authors demonstrated that As(III) was oxidized to As(V) over the
complete time course of the experiment, resulting in removal efficiency of 90%.

A packed-bed column of a continuous flow reactor with Rhodococcus equi cells immobilized on
rice husks was used both to investigate the As(III)-oxidizing performance of the reactor and also
to develop a deterministic mathematical model for explaining the trend of arsenic removal (Bag
et al., 2010). Simulated arsenic-laden water and naturally occurring water with arsenic
concentrations ranging from 50 to 100 ppb were used. The cells were able to detoxify the
simulated arsenic water in the tested range and a maximum As(III) removal efficiency value of
95% was obtained in these processes. Finally, the authors stated that the simulated results were
satisfactorily comparable to the experimental results. Similarly, a modeling analysis of
autotrophic As(III) oxidation in a biofilm reactor using T. arsenivorans strain b6 under different
As(III) concentrations (500–4000 mg/l) was developed (Dastidar and Wang, 2012). The authors
concluded that the As(III) oxidation efficiency rate of the reactor ranges from 48.2 to 99.3% and
the observed and predicted As(III) flux data exhibited good agreement. As(III) oxidation can not
only be performed by pure cultures, but also by bacterial consortia, as reported by several
authors (Michel et al., 2007; Michon et al., 2010). In this papers, the author investigated the
formation and activity of an As(III)-oxidizing biofilm developed by the CAsO1 bacterial

13
consortium in a bioreactor using pozzolana (volcanic material) as growth support. In particular,
the biofilm structure of both a consortium and a pure culture of T. arsenivorans was a physical
barrier decreasing As(III) access to sessile cells and the induction of As(III) oxidase activity
(Michel et al., 2007). Nevertheless, it was indeed pointed out that the efficiency of the reactor
needed to be enhanced by optimizing and controlling several parameters affecting biofilm
formation, such as temperature and the addition of extracellular polymeric substances.
Experiments showed that the CAsO1 consortium was able to oxidize not only in the range of the
‘mg/l order of magnitude’, but also in the concentration scale of the ‘μg/l order of magnitude’
(Michon et al., 2010).

After the biological oxidation of As(III), it is necessary to remove the produced As(V) by using
sorbents. The combined processes of biological oxidation and chemical removal onto synthetic
sorbents in one- and two-step processes have been investigated by several authors. In one of the
first applications of this technique, (Lièvremont et al., 2003). found that two mineral phases,
kutnahorite and chabazite, showed different abilities in adsorbing arsenic after its biological
oxidation and in performing abiotic oxidation. Their results also showed that the studied bacterial
strain performed a fast oxidation of As(III) at high arsenic concentrations in the presence of
chabazite, and that As(V) was efficiently removed by kutnahorite. A two-phase detoxification
process was therefore suggested. By using a mixed culture of heterotrophic As(III) oxidizers,
Arsenic removal by activated alumina was greatly enhanced by bacterial oxidation of As(III) to
As(V), suggesting that the two processes must be performed consecutively for the attainment of
optimum conditions in each step (Ike et al., 2008). More recently, Wan and colleagues set up two
reactors, one filled with sand and cells of T. arsenivorans, and the other with zero-valent iron
(ZVI) (Wan et al., 2010). The process was shown to be successful in terms of biological As(III)
oxidation, and the chemical removal of arsenic was enhanced by ZVI, even though a decrease in
adsorbing power was observed during the biological oxidation peak, probably due to biofilm
formation on the ZVI surface and changes in the physicochemical conditions.

The possibility that arsenic-oxidizing bacteria have a negative effect on arsenic retention by
sorbents can be related to the reactive microbial surfaces and extracellular polymeric substances.
Huang and coauthors highlighted the competitive interactions between phosphate and
carboxylate groups of the cell surface of Shewanella putrefaciens, and As(V) was sorbed to
goethite and ferrihydrite, leading to a mobilization of arsenic (Huang et al., 2011). Conversely,
Kim and coworkers reported only a minimal effect of a mixture of E. coli, Enterococcus faecalis
and Bacillus subtilis on arsenic removal by iron-impregnated, granular-activated carbon (Kim et
al., 2010).

CONCLUSION AND FUTURE PERSPECTIVE

In conclusion, the research concerning abiotic and biotic factors influencing the arsenic behavior
in groundwater is still challenging for health-related issues and bioremediation applications. The

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impact of arsenic-contamination on the environment was widely studied over the last decades
and several studies focusing on arsenic distribution, geochemistry and biological transformations
were performed. However, information specifically related to groundwater is scattered in the
scientific literature and more explicative and clarifying studies are necessary to better elucidate
arsenic-related microbial activities in this environment. Due to the unsatisfactory experimental
conditions tested so far, the potentialities of microorganisms
in arsenic removal processes in natural waters are not fully exploited as well as the diversity and
distribution of functional genes controlling arsenic transformation in such environments.
Therefore, specific studies using experimental conditions mimicking real situations (e.g., long
term studies performed at large scale and/or in water treatment plant) may help to evaluate the
efficiency and the applicability of microbial arsenic remediation processes in combination with
conventional methods.

In addition to curiosity-driven research, the removal of arsenic from contaminated ground-water,

which is often used as a source of drinking water or for irrigation of crops, requires further
research. Although pure culture experiments have shown good results regarding the removal of
arsenic from groundwater, most of the authors believe that large-scale purification can only be
achieved with natural consortia. Pure cultures require sterile bioreactors and expensive
substrates. In addition, these cultures might not be tolerant to the fluctuating composition of
contaminated groundwater. Natural consortia, on the other hand, have been used for decades in
the treatment of municipal waste water; they can be kept in open systems and consist of a suite of
microbes that will be selected by environmental conditions. By tweaking these conditions, the
performance of the process of interest can be drastically improved. So far, the microbes involved
in these processes have been treated as a ‘black-box’, but today, with the use of next-generation
sequencing and other high-throughput techniques, the direct or indirect roles of the
microorganisms and their interactions can be ascertained in detail.

Among the different biological processes for arsenic removal from contaminated groundwater,
microbiological As(III)-oxidation resulted to be the most promising application. The use of
autotrophic As(III)-oxidizers may be preferred since this process is not dependent on organic
carbon for their growth. Nevertheless, also the application of heterotrophic As(III)-oxidation
could be exploited in bioremediation. Several aspects need to be further studied to efficiently
implement oxidizing bio-filters in arsenic treatment units on a long-term basis. They may include
the optimization of both biofilter geometry and hydraulic parameters (e.g., influence of flow
velocity, monitoring of clogging after prolonged use). Moreover, additional aspects deserving
further investigation concern the fine-tuning biofilm development (e.g. control to prevent the
growth of pathogens in the filter, need of carbon supply to support fast reactions with time).
Lastly, coupling of biological and chemical processes must be assessed in order to achieve a
kinetic of biological oxidation compatible with chemical process for sufficient daily production
of arsenic-free drinking water.

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