1. BIOTRANSFORMATION OF MANGANESE:
BIOGENIC METALS IN WATER TREATMENT AND MANAGEMENT
A term paper
by
ZURI DALE
WATER MANAGEMENT AND HYDROLOGIC SCIENCES
Chair of Committee, Raghupathy Karthikeyan
Committee Members, Terry Gentry
Jacqueline Peterson
Head of Department, Ronald Kaiser
May 2013
Major Subject: Water Management and Hydrologic Science
Copyright 2013 Zuri Dale

2. TABLE OF CONTENTS
I INTRODUCTION
1.1. Introduction.................................................................................... 1
1.2. Manganese Chemistry.................................................................... 3
II BIOTRANSFORMATION OF MANGANESE/OXIDE FORMATION
2.1 Manganese Enrichment and Cultivation......................................... 9
2.2 Identification and Detection............................................................ 10
2.3. Pseudomonas putida Mn B1 and GB-1......................................... 11
2.4 Bacillus sp. SG1.............................................................................. 16
III INFLUENCE OF MANGANESE OXIDE ON SCAVENGING AND
ADSORPTION OF TRACE ELEMENTS
3.1. Manganese..................................................................................... 22
3.2. Arsenic........................................................................................... 25
3.3. Selenium........................................................................................ 28
IV. SUMMARY AND CONCLUSIONS
4.1. Biogenic metals in Water Treatment............................................. 31
4.2. Summary and Future Recommendations....................................... 35
V. REFERENCES..................................................................................... 37
VI. CURRICULUM VITAE....................................................................... 41

3. 3
I. INTRODUCTION
1.1 Introduction
Manganese oxide minerals, ubiquitous in soils and sediments, play a key role in
the biogeochemical cycles of metals and organic carbon while influencing significantly
the transport and fate of both contaminants and nutrients in the environment via sorptive,
catalytic, and oxidative processes (Villalobos et al., 2003).
Microorganisms have potential to change the oxidation state of metals while
simultaneously depositing metal oxides onto cellular surfaces. As a result, recent
investigations involving metal-microbe interactions are gaining interest and attention is
being given to the precipitation of manganese and the influence on the
oxidation/reduction of trace elements.
Biologically oxidized Mn oxides are of amorphous, poorly crystalline structure
and as a result possess a large surface area, high catalytic activity, and higher adsorption
capacities (Jiang et al., 2010; Scott and Morgan, 2006; Borch et al., 2009; Nelson and
Lion, 2003). Several studies have focused on the biological synthesis of Mn oxides and
it is currently understood that biological oxidation of Mn occurs several orders of
magnitude faster than chemical oxidation with biogenic Mn oxides having a higher
binding energy per unit surface area than commercially available oxides (Hennebel,
2009). Ulrich, 2007 suggests that adsorption efficiency may be 2 to 5 times higher than

4. 4
that of chemically synthesized Mn oxides and it appears that bacteria are able to produce
a soluble Mn (III) complex of nanoscale with specific surface areas between 98 and 224
m2
/g. Biological Mn oxidation is an important process in the environment, because it not
only controls the availability of manganese itself, but it is likely to exert influence on the
bioavailability of other elements whether toxic or nutrients (Nelson and Lion, 2003).
Adsorption to mineral surfaces generally controls the concentration of most
contaminants and manganese oxides play a major role in mineral adsorption (Zhu and
Schwartz, 2011). By utilizing redox processes to convert a soluble fraction, Mn (II), to
an insoluble fraction (Mn IV), the fate and cycling of manganese as well as other
contaminants may be influenced.
Manganese oxides can adsorb arsenic (As), cadmium (Cd), cobalt (Co), mercury
(Hg), nickel (Ni), plutonium (Pu), uranium (U) ,Zinc (Zn) cations as well as pesticides
among other trace elements (Ulrich, 2007). Furthermore, because this process is
autocatalytic, manganese oxidation allows production at the sites of contamination
making in-situ remediation possible.
In this review, the chemistry of Manganese will be visited as well as the ability
of microorganisms to produce stable biologically produced Manganese oxide structures.
This review will also serve to demonstrate that metal-microbe interactions have
influence on water quality and treatment. Not only are these oxides able to remove
soluble manganese from aqueous systems, but, moreover, this biologically synthesized
manganese oxide catalyst may be used to control the cycling and bioavailability of other
trace elements including Selenium and Arsenic.

5. 5
1.2 Manganese Chemistry
Manganese is the 5th
most abundant transition metal, second only to iron as the
most common heavy metal, and an important trace element in soils and aquatic
environments (Post, 1999; Tebo et al., 2004; Ulrich, 2007). Manganese is found
primarily in the earth’s crust and is released from metamorphic rocks by surface water
interactions at the earth’s surface. Manganese is an abundant metallic element and
constitutes about 0.1% of the earth’s crust.
The elemental form of Manganese does not occur very naturally in the
environment, but Mn is a component of in excess of 100 minerals. Manganese oxide,
manganese carbonate, and manganese silicate are a few of the most common mineral
forms (Kohl and Medlar, 2006). Soluble Mn (II) is highly mobile in aqueous systems
and in the presence of oxygen, Mn (II) is readily oxidized to Mn (IV) forming more than
30 oxide/hydroxide minerals (Granina and Callender, 2006; Ulrich, 2007). This array of
minerals is primarily due to manganese occurring naturally in numerous oxidation states,
which subsequently produce a wide range of multivalent phases. Among the various
oxidation states in which manganese exists, the II, III, and IV oxidation states are of the
most biological significance (Post, 1999). Manganese oxides are characterized by having
open crystal structures, large surface areas, and high negative charges (Tebo et al., 2004;
Ulrich, 2007).

6. 6
In the natural environment, manganese oxide minerals commonly occur as fine-
grained aggregates and coatings with the most extensive deposition of Mn oxides
occurring in the oceans as nodules. The nodules are most abundant in oxygenated
environments with the source of Mn thought to be continental runoff and hydrothermal
volcanic activity at mid-ocean spreading centers (Post, 1999). Though there has been
debate concerning the direct mechanism of nodule growth in ocean waters, it is now
understood that bacteria may serve as catalysts for Mn oxide precipitation. Figure 1
presents an idea of the relative distribution and abundance of manganese in nature
(DePalma, 1993).
Figure 1. Relative distribution of Mn in the natural environment (DePalma, 1993).
Thermodynamically, under aerobic conditions, in the absence of oxygen, Mn (II)
is favored at low pH, whereas in the presence of oxygen at higher pH, Mn (III) and Mn
(IV) are favored with a negative free energy of approximately 16 kcal/mol (Tebo et al.,

7. 7
2004; Nealson, 1992) (Figure 2). Mn (II) has a very large activation energy which
allows it to remain stable in aquatic environments. However, this activation energy
barrier may be overcome by raising the pH or catalysis by microorganisms.
Mn (II), which appears most often in solution or absorbed to minerals, is usually
soluble as an ion or complexed to organic or inorganic ligands. Mn (III), unless
complexed to ligands or incorporated in enzymes, is thermodynamically unstable in
aqueous environments and readily disproportionates to Mn2+
and MnO2 (Brouwers et al.,
1999; Tebo et al., 2004;). Mn (IV) occurs in insoluble oxides and hydroxides.
Figure 2. At low oxygen and low pH, Mn (II) predominates. In more oxygenated, high
pH environments, Mn (III) and Mn (IV) predominates (Tebo et al., 2004, Ulrich, 2007).

8. 8
II. BIOTRANSFORMATION OF MANGANESE
There was once widespread debate concerning whether manganese oxidation in
nature was primarily chemical or biological. It is generally agreed now to be a
biological occurrence (DePalma, 1993). Bacteria gain energy and reproduce by
mediating the transfer of electrons from compounds that readily donate electrons to
compounds that readily accept electrons (Brown, 2007). As compounds are oxidized or
reduced, they are often converted to different, often innocuous compounds that are often
more thermodynamically stable than the original compound (Brown, 2007).
Bacteria are able of oxidizing Mn(II) to Mn(III) and Mn(IV) enzymatically and
the kinetics of this reaction are a lot faster than the abiotic reaction, with a half life in the
order of days compared to hundreds of years (Ulrich, 2007). Many assumptions have
been made concerning why bacteria oxidize Mn (II), however there is very little known
about this phenomenon. Though there is no supported evidence, one possibility is that
bacteria oxidize Mn (II) for ATP generation (Ulrich, 2007). And while this reaction is
thermodynamically favorable, there is no direct evidence linking oxide formation to
energy conservation.
Microorganisms absorb trace metals by producing extracellular polymers that
have well established binding properties, therefore, alternate suggestions have been that
bacteria utilize Manganese as protection from reactive oxygen species, UV radiation,
predation, heavy metal toxicity and other oxidants in the environment.

9. 9
Oxidation of manganese is known to undergo catalysis by a number of
microorganisms including bacteria and fungi and Mn (II)-oxidizing bacteria have been
identified in a growing number of divergent phylogenetic lineages in the bacterial
domain, such as Firmicutes, Proteobacteria and Actinobacteria (Tebo et al., 2005; Ulrich
2007) Figure 3.
Figure 3: Phylogram of the domain bacteria showing representative Mn (II)-oxidizing
bacteria (Tebo et al., 2005).

10. 10
Bacteria that are able to oxidize Mn are referred to as Manganese Oxidizing
Bacteria (MOB) and four model organisms have been isolated in pure culture and
demonstrate an ability to catalyze this reaction. This broad phylogenic diversity seen in
Figure 3 reflects the diversity seen the more extensively studied model organisms.
These species include Pseudomonas putida Mn B1 and GB-1, Bacillus sp. SG1, and
Leptothrix discophora. The Mn-oxidizing bacteria present in source water can grow and
produce under appropriate conditions, and oxidize Mn (II) leading to precipitation of the
oxidized form, Mn (IV) (Jiang et al., 2010).
Though, these species are phylogenetically diverse; they all require enzymes
homologous to the multi-copper oxidase enzyme for Mn oxidation (Hennebel, 2009;
Tebo et al., 2004). An overview of Pseudomonas putida Mn B1 and GB-1 and Bacillus
sp. SG1 will be presented further in later sections. Leptothrix discophora will not be
discussed in further detail as it was not an original consideration of our laboratory
studies.

11. 11
2.1. Manganese Enrichment and Cultivation
As long as Mn toxicity is evaded, Mn-oxidizing bacteria can be obtained in pure
culture and grown in either liquid or solid media. A number of studies have
demonstrated the growth of Mn-oxidizing bacteria but there is a general consensus that
the following, among other factors, influence the growth and cultivation of Mn oxidizing
bacteria.
A.) pH- Manganese oxidation is strongly pH dependent. The addition of a buffer to
growth medium will serve to prevent abiotic oxidation that occurs at higher pH
values. Jiang et al., utilized HEPES buffer in a characterization study performed.
HEPES is one of the twelve GOOD’s buffers and is used largely at maintaining
physiological pH despite changes in carbon dioxide (produced by cellular
respiration) concentration when compared to carbonate buffers.
B.) Carbon Source- Simple carbon sources are generally used in Manganese
oxidation as oxidation normally occurs after growth has slowed or ceased.
(Nealson et al., 1992)
C.) Yeast Extract- Water soluble portion of autolyzed yeast and an excellent
stimulator of bacteria and cell growth. Rich in vitamins, minerals, and digested
nucleic acids.
D.) Casamino Acids- Supplies a hydrolyzed protein source. Mix of amino acids used
to supplement growth media.

12. 12
E.) Mn- The source of Manganese may vary. Different studies have utilized
carbonates, chlorides and sulfates and it appears that the source of Mn may have
little influence on the oxidation products. Several studies mention however, that
filter-sterilization of Manganese is advisable as opposed to autoclaving as auto-
claving may partially oxidize the Mn source. This would lead to difficulty in
determining if the reaction is biotic or abiotic. In the natural environment, Mn
concentration rarely exceeds 1-5 micromolars. (Nealson et al., 1992). Under
laboratory conditions, concentrations above this level may be toxic to bacteria.
2.2. Identification and Detection
Visually, Manganese oxidizing bacteria and Manganese oxides are identified by the
presence of a brownish-black precipitate, however more in depth determination is
usually needed. Quantifying the partitioning of Mn between soluble and particulate
fractions is a major component of nearly all laboratory or field biotransformation
research (Tebo et al., 2007). Although there are multiple spectrophotometric techniques
for measuring oxidized forms of Mn, the preferred method is the leucoberbelin blue
method (LBB) as will be discussed.
Leukoberbelin blue (LBB) is a colorimetric determinant of Manganese oxide
which upon interaction with Mn (III) or Mn (IV) is quantified by reaction with reductive
dye that yields a blue color. The intensity of the coloration is a function of the amount of
Manganese oxides reduced (Jiang et al., 2010) Color intensity is measured using a

13. 13
spectrophotometer. A benefit of the LBB method as opposed to alternate methods is that
in there is less interference from Fe.
LBB is prepared by preparing .04% LBB in 45mN acetic acid dissolved
overnight and stored at 4°C protected from light. This is then stored in the fridge for at
least 24 hours prior to first use. LBB will oxidize slowly while stored. Benefits of use
include that a stock solution may be kept for up to one year. An aqueous solution of
potassium permanganate was utilized in Tebo et al., 2007 to obtain a standard curve due
to its strong oxidizing capabilities in acidic solutions by which manganese oxidation is
compared.
2.2. Pseudomonas putida Mn B1 and GB-1
Pseudomonas manganoxidans encompasses the species of bacteria that are
differentiated by their ability to oxidize manganese. Pseudomonas manganoxidans is
more commonly identified as belonging to the common species Pseudomonas putida.
Historically, bacterial identification is a difficult process, and unfortunately not much
attention is given to the tedious, time-consuming testing that is needed to identify new
strains of bacteria. However, in 1873 Schweisfurth attempted this feat by examining
numerous sites that contained manganese oxide deposits. From these sites he was able to
identify approximately 200 strains that formed brown colonies on nutrient agar. After
repeated testing, thirty of these strains retained the manganese oxidizing phenotype. The
assignment of temporary names to these clusters was attempted and literature describes

14. 14
that by 1992, Pseudomonas manganoxidans was referred to as just Pseudomonas putida.
Currently this is the ATCC (American Type Culture Collection) catalogue designation.
Pseudomonas putida is a fresh-water, aerobic, heterotrophic species of which
two oxidizing strains have been identified. Oxidation of Manganese is similar in both
strains and multiple studies suggest that the most activity occurs in the stationary/late
stationary growth phase at the extracellular apparatus (Brouwers et al., 2000; Tebo et al.,
2004) Several studies also suggest that in both strains oxidizing activity appears to be
dependent upon oxygen concentration present in the cultures during growth. Okazaki et
al., 1997 demonstrated specifically that in strain GB-1, cellular oxidizing activity
doubled with oxygen increases from 20 to 30% saturation. This particular study also
presented the idea that oxidizing activity decreased at higher oxygen concentrations
suggesting that the oxygen concentration in the medium at the late logarithmic phase
strongly influences the effect on the amount of Mn-oxidizing factor produced by the
bacteria in the early stationary phase. This information provided great insight into
relationship between oxygen concentration and oxidizing activity, however I was unable
to find any additional literature to support this.
Jiang et al., 2010 also investigated the Manganese oxides produced by
Pseudomonas putida MnB1 in an effort to provide insight into the growth kinetics of the
microorganism. This study demonstrated, consistent with previously mentioned
understood mechanisms of metal-microbe interactions, that oxides are deposited on
cellular surfaces. This is a stronger indication that in both strains the oxidizing apparatus
may be located at the extracellular membrane.

15. 15
Jiang et al., 2010 also suggested that in Pseudomonas putida Mn oxidation was
both a temperature and pH dependent reaction. This study also concluded that Mn
oxidation also occurs in the late stationary phase but with maximum oxide generation
occurring at neutral pH with the rate of oxidation decreasing as pH decreased (Figure 6).
This information is in agreement with other studies that suggest biogenic oxidation
Pseudomonas putida is inhibited at pH less than 6. Result of pH and temperature studies
conducted in Jiang et al., are presented to demonstrate this phenomenon.
Figure 6. Rate of Mn (II) oxidation as a function of initial pH. (Jiang et al., 2010.)
A sharp temperature optimum is an also effective indicator that a geochemical
process is mediated by biology and it has been used to demonstrate that enzymes
catalyze Mn oxidation in natural samples (Tebo et al., 2004). Jiang et al., 2010 further

16. 16
provided evidence that Pseudomonas putida was temperature sensitive with optimum
oxidation rate occurring at temperature 24 degrees Celsius (Figure 7).
Figure 7. Rate of Mn (II) oxidation as a function of temperature. (Jiang et al., 2010.)
Further literature review suggests that both strains MnB1 and GB-1 are subject to
genetic manipulation. Transposon mutagenesis has been used to isolate mutants
defective in their ability to oxidize manganese (Brouwers, et al., 1998) In fact, in a 1999
Brouwers et al. study GB-1 actually lost its ability to oxidize manganese by transposon
insertion Figure 8. While genetic investigations are out of the scope of this review, it is
important to note that this phenomenon is possible, thereby providing some insight into
potential reasons why our laboratory strains failed to produce manganese oxide after a
period of time.

17. 17
Figure 8. Insertion of transposon genes in P. putida GB-1 provide a phenotypic
indication that of an lack of oxidizing ability. Colonies that were once brown (left) are
now non oxidizing white (right) colonies when stained on agar plates. (Brouwers, et al.,
1999)
Toner et al., 2006 also investigated the adsorption of Zn to Mn oxides with a
biofilm produced by P.putida Mn-B1. The maximum adsorption to the oxide was .37
mol of Zn per mol of Mn oxide. This particular study went on to describe that Mn oxides
also demonstrated sorption capacities for Ni and Co and that adsorption efficiency was
approximately tenfold higher than that of chemically synthesized oxides (Hennebel et
al., 2009; Toner et al., 2006)

18. 18
2.3. Bacillus sp. SG1
Bacillus sp. SG-1 is a gram positive marine organism that forms inert spores
upon nutrient limitation and it is the spores of Bacillus promote that Manganese
oxidation (Brouwers et al., 2000). The outmost layer, the exosporium, is where oxidizing
activity is localized. Because the spores of Bacillus are inert, their oxidizing capacity is
not straightforwardly related to metabolic function. In fact, Bacillus is one of the only
strains in which germination reduced oxidation in vegetative cells. Interestingly of
Bacillus sp. SG-1, it is able to either oxidize or reduce depending upon its life stage.
Hastings and Emerson, 1986 conducted a study exploring the Manganese oxides
formed by marine Bacillus sp. SG-1. Since this organism is a marine species,
experimental conditions were maintained near that of seawater in order to yield realistic
result. The bacterium was isolated from a near shore culture where spores of that culture
had been shown to bind and oxidize manganese at pH values below which manganese
normally auto-oxidizes. In this study oxidation facilitated by the spores was four orders
of magnitude greater than abiotic oxidation.
In a 1986 study by Vrind et al. spores were completely able to remove Mn (II)
from K-Medium with spores bound within one hour demonstrating that by using high
spore concentrations, oxidation time could be reduced from days to hours. Both of these
mentioned studies demonstrated however that there is a decrease in oxidizing activity as
the amount of manganese oxide increases. Based upon understood mechanisms of

19. 19
enzyme- substrate interaction, it is likely that decreased accessibility of active sites is
responsible for this.
In Bacillus sp. SG-1 it is thought that oxidation occurs only after Mn (II) is
bound, so this reaction is likely a two-step process. First, adsorption followed by proton
release and subsequent rapid oxidation of adsorbed Mn. Nealson and Rosson, 1992
presented similar findings in their study using dormant spores of precipitated Mn with
the amount oxidized being a function of spore concentration not necessarily Mn
concentration.
In considering whether reports of these finding hold environmental relevance,
two questions are posed. Are the properties of spores consistent in nature AND are the
nature concentrations of spores ever high enough to yield significance? In answer to the
latter question, it appears that binding affinity of spores is sufficient to be useful in all
marine environments as multiple studies investigated binding over a range of
concentrations.
The use of Bacillus in laboratory investigations is promising. It is well
established that the spores may bind Mn (II) which may further catalyze oxide
production. And as previously stated, the autocatalytic nature of this process continues to
provide geochemical implications (Nealson and Rosson, 1992).

20. 20
III. INFLUENCE OF MANGANESE OXIDE ON SCAVENGING AND
ADSORPTION OF TRACE ELEMENTS
Redox processes control the availability, toxicity, and mobility of several major
and trace elements and it is understood that redox transformations in terrestrial
environments can be used to control water quality (Borch et al., 2009). The redox
properties of Mn make it central to a variety of processes and result in significant and
often rapid biogeochemical cycling that is mediated by biotic oxidation, biological
uptake and mineral formation (Tebo et al., 2007).
Although it has been known for over a century that microorganisms have the
potential to change the oxidation state of metals, it was only during the past few decades
that researchers realized that these processes present new applications. These include not
only the treatment of drinking water but the removal of recalcitrant pollutants under
anaerobic conditions, the generation of electricity out of sediments and wastewater
streams, metal recovery in combination with the formation of novel biocatalysts and the
remediation of metal-contaminated soils and wastewaters (Brown, 2007, Hennebel,
2009).
Mn is an element whose distribution and chemical speciation is kinetically
controlled, thus allowing for the intervention of microbes and microbial products into the
system (Nealson, 1992). A few of the ways in which bacteria may oxidize manganese
are presented in Table 1.

21. 21
Table 1. Potential mechanisms of oxidation by bacteria (Nealson, 1992)
Manganese oxidizing bacteria can be supplied isolated from virtually any habitat.
It has been observed in field investigations that habitats with elevated levels of Mn also
have elevated levels of Mn-oxidizing bacteria, however in certain cases there are habitats
in which manganese oxidizing bacteria can isolated and identified solely by morphology.
A common feature of these habitats in which these field investigations are
conducted is a constant Mn supply. This supply can be provided usually under one or
two circumstances: 1. Oxic /Anoxic interfaces (Figure 8) and 2. Input of anoxic water
into aerobic environments (Nealson, 1992) (Figure 9).

22. 22
Figure 8. Manganese (II) Input Option One: Redox Interface (Nealson, 1992)
Figure 9. Manganese (II) Input Option One: Input of Anaerobic Water (Nealson, 1992)

23. 23
Biological water treatment processes rely on the growth of bacterial populations
capable of mediating redox reactions. Several studies have focused on the biological
synthesis of Mn oxides with biological oxidation of Mn occurs several orders of
magnitude faster than chemical oxidation (Hennebel, 2009).
The characteristics of high specific surface area and the presence of a bacterial
carrier matrix make biogenic oxides useful as oxidants and reductants. Next to oxygen,
Mn oxides are some of the strongest naturally occurring oxidizing agents in the
environment (Tebo et al., 2005). Not only do Manganese oxides participate in a variety
of redox reactions, but also may serve as terminal electron acceptors for bacterial
respiration.
As mentioned previously, the environmental fate and behavior of toxic transition
metals are governed by interactive biogeochemical processes, such as adsorption,
complexation, and multiple biological interactions (Nelson and Lion, 2003). The
following section will focus on Manganese, Arsenic and Selenium and the influence of
Manganese oxide on their oxidation products.

24. 24
III.1 Manganese
Manganese occurs naturally in food, soil, air, and water at low levels. Deutsch et
al., 1997 even found Mn concentrations ranging from 0.3 to 11.3 ug/L in rain and snow
samples in Germany. In recent years the utilization of heavy metals such as manganese
by fertilizers, mining, and petrochemical industries has increased dramatically. Suffice it
to say, that Manganese is everywhere.
Though manganese is generally considered a secondary hazardous element,
dissolved manganese can serve as a nuisance in a water supply and result in reduced
acceptance by consumers (WHO, 2004). At concentrations exceeding 0.1 mg/L, the
manganese ion imparts an undesirable taste to beverages and stains plumbing fixtures
and laundry (Griffin, 1960). At concentrations as low as 0.02 mg/L, manganese can form
coatings on water pipes that may later slough off as a black precipitate (Bean, 1974). A
number of countries have set standards for manganese of 0.05 mg/L, above which
problems with discoloration may occur (WHO, 2004) though concentration at or even
below the USEPA Secondary Maximum Contaminate Level can create drinking water
problems.
Historically, in water supplies Mn was considered to be solely an aesthetic issue,
however in the early 1990’s reports began to generate suggesting that Mn in drinking
water may also have illness causing effects. By 1998, Mn was included on USEPA’s
Drinking Water Contaminate Candidate List (DWCCL) as a regulatory determination
priority contaminant based on the finding that it may have adverse health effects on the
health of persons.

25. 25
Manganese deposits have potential to cause widespread problems of poor
aesthetic quality (brown-black discoloration), staining of fixtures, equipment, swimming
pools, and laundry (Sly et al., 1989). In areas where oxygen content is low, the
manganese-bearing water is clear and colorless (WHO, 2004), however, when
manganese (II) compounds undergo oxidation, manganese is precipitated and forms
particulates that may then be settled out of the water. Household problems begin to occur
with manganese in the precipitated form. If manganese remains soluble it is usually able
to pass through potable water undetected, but Manganese does not remain dissolved
while in our water distribution system prior to reaching a consumers home. And most
oftentimes clothing is bleached thereby by oxidizing manganese imparting discoloration
on clothing.
The presence of manganese in raw water presents problems for water treatment
authorities because unlike iron, manganese is not chemically oxidized by air at neutral
pH nor is it removed during water treatment processes unless a chemical oxidation step
is included (Sly et al., 1990). During the summer, Mn can be a problem in municipal
water supplies and public and private wells because during this time the oxygen level
falls and reduction of Mn oxides is promoted.
At once the presence of Manganese in drinking water was initially a groundwater
problem. There was a time when the simple solution included digging an alternate well
or diluting the water containing the Manganese. However, as a result of increasing
demand more treatment became required. Early very limited understanding of
Manganese control led treatment facilities to believe that the issue of Manganese was not

26. 26
a major one. At a time, many treatment plants utilized granular media coated with a
chlorine oxidant. For a while, this process was sufficient at manganese removal but most
treatment facilities were not even aware that this was occurring.
As time has progressed the water treatment industry has changed with stricter
laws concerning disinfectant by-product control to reduce the use of chlorine as an
oxidant (Kohl and Medler, 2006). Because of this, the once seemingly small amount of
Manganese once present became important as treatment began to eliminate granular
filtration media with chlorine as an oxidant.
Manganese is present in drinking water in three forms which include particulate,
colloidal and dissolved form (Kohl and Medlar, 2006). Particulate form includes large
oxidized material while colloidal form includes smaller oxidized material with the
dissolved form being soluble. Particulate manganese removal is typically successful
through filtration processes however colloidal manganese still has the ability to pass
through filters. In essence, all soluble manganese must be converted into insoluble form
and removed by filtration with dissolved oxygen levels and microbial activity principally
determining which chemical form predominates.
Microbiological treatment has been used to precipitate soluble manganese from
water supplies, especially in Europe (Griffin, 1960; Mouchet, 1992, DePalma, 1993).
Peitchev and Semov, 1988 described naturally-occuring manganese-oxidizing bacteria
colonized sand filters through which Mn2+
-containing water is passed, resulting in near-
complete removal of soluble manganese.

27. 27
III.2 Arsenic
Arsenic is a ubiquitous element that ranks 20th
in abundance in the earth’s crust,
14th in the seawater, and 12th in the human body (Mandal and Suzuki, 2002). Arsenic is
comprises about five hundred–thousandths of 1% ((0.00005%) of the earth’s crust and
naturally occurs in over 200 different mineral forms, of which approximately 60% are
arsenates, 20% sulfides and sulfosalts and the remaining 20% includes arsenides,
arsenites, oxides, silicates and elemental arsenic (As) (Kramar and Suzuki, 2002).
Arsenic contamination is one of the most problematic environmental issues, with
the presence of arsenic in groundwater posing the largest environmental threat to
humans (Hennebel et al., 2009 ). Arsenic is highly toxic and there is no evidence of a
benefit of intake.
The chemistry of arsenic in aquatic systems is quite complicated as the element
may exist in any one of the four stable oxidation states (+5, +3, 0, -1 and -3)
(Chakravarty et al., 2002). However, in natural waters arsenic normally occurs in the
oxidation states +III (arsenite) and +V (arsenate) with the removal of As (III) being more
difficult than the removal of As (V) (Bissen and Frimmel, 2003). It is understood that As
(III) is more toxic than As (V). Arsenate (III), the more toxic form, persists in aerated
water, even at high pH, but is easily oxidized by manganese oxides (Driehaus et al.,
1995). Therefore, As (III) has to be oxidized to As (V) prior to its removal.
Arsenic concentrations in groundwater are usually below 5 ug/l, but geochemical
mobilization, contaminated soils in industrial regions, and the use of arsenic pesticides
result in elevated concentrations in the environment (Driehaus et al., 1995). The drinking

28. 28
water standard of Arsenic is 0.05 mg/l but new information on the toxicity of Arsenic
has lead to discussions of lowering this standard.
A number of methods have been employed to remove Arsenic from water
systems, however a truly efficient removal process will result in total conversion of As
(III) to As (V). The oxidation of arsenic in the presence of air or pure oxygen is
typically slow, however this reaction may be accelerated with the used of advanced
oxidative processes such as manganese oxide coated sands and/or direct microbial
oxidation.
Most of the commonly used Arsenic removal techniques fail to adequately
remove As (III). Clifford et al., 1983 observed that only a small percent of As (III) was
oxidized in the presence of air and only 25% of As (III) was oxidized when purging a
solution initially containing 200 μg/L As (III) with air. Similarly, Frank and Clifford,
1986, reported that only 8% of Arsenic was oxidized in solution within 60 minutes. The
coagulation precipitation technique is not as efficient in As (III) removal as it is As (V)
removal and external oxidizers tend to depreciate drinking water quality. Suffice it to
say, that there has been a need for inexpensive arsenic removal technology for some
time. Chlorine has proven to be effective and reliable, but as previously mentioned by-
products are produced. Moreover, chlorine is not used as an oxidizing agent in some
counties such as Germany.
Manganese oxides are capable of oxidizing arsenite, and are well described in
literature. Hambsch et al., 1995 showed that bacteria can oxidize As (III) in the presence
of at least 1 mg/L of O2 and if manganese is present in groundwater, the presence of

29. 29
bacteria may oxidize arsenite without the use of any additional treatment steps.
Chakravarty et al., 2002, described arsenic removal from groundwater utilizing a
ferruginous manganese ore. This study investigated adsorption capability in six
groundwater samples and in each case Arsenic removal was almost 100%.
Manning et al., 2002 demonstrated in a study that at neutral pH conditions the
oxidation of arsenite by manganese oxide resulted in an increase in arsenate. This study
further described that in the process of oxidation, an alteration of the extracellular
manganese oxide surface occurs, that allows further absorption of As (V). Overall data
in this study suggested that the manganese oxide produced arsenic removal rates of more
than 90%.

30. 30
III.3 Selenium
Selenium is in group 16 on the periodic table, and though it has properties of
both metals and nonmetals, it is generally described as a nonmetal and ranks 70th
in
abundance of naturally occurring elements. Despite selenium being needed in trace
amounts, the range of intake leading to deficiency and toxicity in humans is extremely
narrow and shows properties of being a nutrient in small quantities and toxin in high
concentrations (Goldberg et al., 2008). The WHO guideline for selenium in drinking
water in the United States is 50ug/L-1
(Plant et al., 2004)
Selenium is needed by both humans and animals in trace amounts and lack of
selenium in the body results in immune suppression. Low dietary intake of selenium
poses a risk factor for cancer while prolonged exposure has deleterious effects (Brouwer
et al., 2000). The primary sources of selenium in water are likely to be sulfides or metal
oxides containing absorbed selenium, especially Se (IV), and the principal concerns of
selenium toxicity are nervous and disruptive system disruption and hair and nail loss in
humans (Plant et al., 2004)
Selenium exists is multiple oxidation states (-II, 0, IV, VI) which contributes to
the complex chemistry of this trace element and deems it redox sensitive (Scott and
Morgan, 2006). In most natural systems, selenium is found primarily in oxidized forms,
selenite (IV) and selenate (VI ) as inorganic oxyanions which exhibit various degrees of
affinity for metal oxide surfaces (Scott and Morgan, 2006). Se (0) and Se (-II) are
virtually insoluble in water (Plant et al., 2004).

31. 31
Historically, analysis for Selenium has been difficult because environmental
concentrations are naturally low. However, the affinity of oxides for selenium has been
documented. In 2006, a study was conducted to experimentally demonstrate that metal
oxide surfaces do in fact influence the behavior and oxidation product of Selenium. Scott
and Morgan, 2006, investigated the rates and mechanisms between Se (IV) and
Manganese oxide indicating that Se (IV) is in fact oxidized into Se (VI) as a result of
adsorption and electron transfer reactions. (This study only sought to be a speciation
study, as further research suggest that Se (VI) is more toxic than Se (IV).) Though this
study investigated synthetic manganese oxide, this study demonstrated experimentally
that redox-active metal oxide surfaces play a role in determining the environmental
behavior of selenium (Scott and Morgan, 2006.)
Interestingly, literature specifically addressing the oxidation of Se (IV) to Se (VI)
by microorganisms is very limited, however reduction studies are very well described. In
contrast to arsenate, toxicity information of selenate is scant. Generally, Se (VI) has the
highest solubility and therefore the most mobility in aqueous systems. In comparison, Se
(IV) is soluble but has a greater affinity for adsorption to soil particles therefore is less
mobile. It appears that even despite minimal evidence of higher toxicity in selenate, all
forms should be considered toxic.
Selenate is only weakly adsorbed by oxides at near-neutral pH, therefore the
oxidation of Se IV to Se VI may serve to enhance selenium mobility in natural waters
(Plant et al., 2004). While this review sought to describe oxidation as a primary removal
technique, microbial reduction is also possible. This does not serve to negate oxidation

32. 32
as a removal strategy but the elemental behavior of selenium does deem that microbial
reduction processes are necessary.
Maiers et al., 1988 demonstrated that that in 44 samples collected from a
reservoir, reduction was observed in 4% of water samples, 100% of soil samples and
92% of sediment samples. A 100 mg/liter reduction of selenate was completed within
one week with a 75 mg/l reduction beyond selenite to a red particulate after 90-95% of
selenate reduction occurring.
Oxidation of elemental selenium was also reported in a 1981 study. Selenium
was oxidized to selenite however trace amount of selenate were also formed although the
amount represented less that 1% of the original amount of selenite formed. These results
are interesting in that two separate microbial studies stated that selenium was oxidized to
selenate with no mention of selenite formation (Sarathchandra, and Watkinson, 1981;
Sapozhnikov, 1937 and Lipman and Waksman, 1923).
Overall selenite/selenate oxidation and reduction is possible but it appears that
efficient treatment methods may only remove selenium if the selenium occurs as selenite
(Maiers et al.,1988). Biologically synthesiseized Manganese oxides may reduce selenate
to selenite with subsequent filtration of the precipitate.

33. 33
IV. SUMMARY AND CONCLUSIONS
4.1. Biogenic metals in Water Treatment
Though they are poorly understood, biological processes have been used since
the 1800s in the form of slow sand filtration and possibly as early as 4000 B.C.E. Until
recently, the biological treatment of drinking water was limited particularly in the United
States, but recent developments may mean that biological treatment of drinking water
may become more feasible and more likely to be accepted by the public (Brown, 2007).
Well designed biological treatment poses no dangers to the safety of drinking
water, and can serve to provide a healthy alternative to treating manganese and other
trace contaminates in drinking water and aqueous systems. Biological processes require
minimal to no chemical usage, low operation and maintenance costs, small scale
operation, high water recovery rates, and the destruction rather that the sequestration or
concentration of contaminants (Brown, 2007). Additionally, increasing demands for high
quality drinking water result in the need for treatment technologies that are capable of
meeting these demands in a cost-effective manner.
Chemical oxidative processes, which are widely utilized, tend to pose adverse
health effects. For example, if chlorine is used as the oxidative agent, residuals may
remain in the treated water. Moreover, for example, chlorination will not reduce Mn
deposition when excessive Mn (II) continues to enter the distribution system. In a
treatment plant, chlorination could considerably worsen oxidation and distribution
causing more serious issues of dirty water (Kohl and Medlar, 2006). Potassium

34. 34
permanganate is also an effective chemical oxidant , however potassium permanganate
is poisonous as well as a skin irritant and itself must be completely removed from treated
water. Suffice it to say that chemical oxidation requires careful continued monitoring
and maintenance.
Biological drinking water treatment will address many drinking water needs and
provide a healthy alternative to chemical treatment processes. As mentioned previously,
this technology is based on the ability of microorganisms – specifically non-pathogenic
bacteria to efficiently catalyze the biochemical oxidation or reduction of drinking water
contaminants and produce biologically stable water.
Various forms of biological treatment processes are used to degrade
contaminants but operate as fixed systems that possess a medium of support upon which
bacterial populations attach. Reactors may either by inoculated with bacterial
communities or water in need of treatment may be acclimated with microorganisms.
In fixed bed biological processes a biofilm exists upon a bed of media with the
media bed being contained in either a pressure vessel or an open basin. Water in need of
treatment may be pumped up or down across this media bed. Fluidized bed processes
also utilize a media that supports the growth of microorganisms, however water is
pumped at an extremely high rate to fluidize the granular media bed (Brown, 2007).
Numerous configurations of biological treatments are available for use and any
well designed system will pose no adverse impact of the safety of water as well as
persons consuming. Additionally, these processes are able to operate on both small and
large scales under a wide range of operating conditions as well as condition of the water

35. 35
being treated. Despite the contaminants discussed in this review, biological processes
are
able to
influence a variety of contaminants are presented in Table 2.
Table 2. Contaminants subject to biological treatment. This table was taken from
(Brown, 2007), however it was complied based upon extensive literature review.

36. 36
4.2 Summary and Future Recommendations
The oxidation of Manganese can be can be catalyzed by microorganisms with
their systems operating in many respects. Whether being facilitated by spores as in
Bacillus or extracellular membranes as in Pseudomonas putida, strong evidence supports
biological oxidation in water treatments. However in order to understand certain
mechanisms or oxidation, multiple things are yet to be understood.
All results verify the amorphous structure and increased surface area of biogenic
oxides, but further experiments are needed to understand the impact on contaminant
dynamics. Moreover, literature lacks information that would expand our general
understanding of the reasons why microorganisms have evolved in this way without any
apparent benefits. Additionally, studies further describing oxidative processes in fungi
would be useful.

37. 37
All of the studies presented describe oxidizing activity under controlled
laboratory conditions. As a result, in all cases more studies are needed that incorporate
more environmental parameters that will better represent field conditions. And while
throughout the course of my literature review organic contaminants were briefly spoken
about, more investigations into oxide interactions with organic compounds such as
endocrine disruptors would be valuable.
In my own research, Pseudomonas putida was investigated. However, overtime,
our strain ceased to produce manganese oxides which limited our ability to produce
reliable results. Future investigations could be pursued in many ways, but my personal
experience is that work is needed in the area of manganese oxide mutagenesis. My
personal limited understanding of this phenomenon did not yield the experimental results
I sought, however I am convinced that under appropriate laboratory conditions
replication studies similar to Jiang et al., 2010 are possible in our laboratory.
Though biogenic Mn oxides have well demonstrated their uses and advantages
insight is needed to synthesize these structures in large enough amount to be used in
water treatment. Pseudomonas putida is well describes as a Mn oxidizing organism but
large-scale studies are lacking. And while this technology is evolving, biological
production does sometimes take days, while chemical oxidation only needs minutes or
hours.
A municipal water supply system has a responsibility to provide water that is safe
for human consumption, with adequate quality at a reasonable price. Biological
processes offer potential additional options to relatively costly, and in some cases

38. 38
ineffective, physicochemical treatment processes for removal of organic contaminants,
nitrogen species, iron, manganese and other trace elements (Kohl and Medler, 2006).
This review sought to present Manganese chemistry and insight into Manganese
oxidizing organisms. Several studies were described validating the use and efficiency of
microorganisms in influencing the oxidation products of certain contaminants. Despite
this, this review has only scratched the surface of what is known and what remains to be
known, but it can be agreed that these processes are efficient and sustainable and that the
use of these treatments are likely to continue to expand.
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43. 43
VI. CURRICULUM VITAE
Zuri Dale
P.O. Box 8344
Houston, Texas 77288
(832) 293-7806
zurielle16@yahoo.com
EDUCATION:
B.S. Biology Texas Southern University May 2011-Cum Laude
MWM. Texas A&M University Expected: August 2014
Current GPA: 3.68
SCHOLASTIC HONORS/AWARDS:
Bridge to Doctorate Fellow/NSF-August 2011-May 2013
Excellence in Research Award 2011
Texas Space Grant Scholarship Recipient 2010
Thurgood Marshall Scholar 2010
Excellence in Research Award 2009
McNair Scholars Program June 05/2009-05/2011
Science Technology Enhancement Scholarship Program (STEP)-08/2007-05/2011
Houston Livestock Show and Rodeo Scholarship 08/2007-05/2011
Worthing Scholarship for Academic Achievement08/2007-05/2011
Frederick Douglass University Honors Program Scholar 08/2007-05/ 2011
RESEARCH PRESENTATIONS and CONFERENCES
Dale, Z., Thomas, R.Galvan, G., 2005 “Characterization of Volatile Organic Compounds and E.Coli in
Urban Watersheds”
• 2004-2005 TWWA / WEAT(Texas Water Works Association/Water Environment
Association of Texas) Grand 1st
place Special Award in Environmental Science
• 2004-2005 Environmental Protection Agency Award for Outstanding Project in the
Field of Science
Dale, Z., Thomas, R. Galvan, G., 2006 “Identification of Trace Metals in City of Houston Fire
Departments”
• 2005-2006 TWWA / WEAT(Texas Water Works Association /Water Environment
Association of Texas) Grand 1st
place Special Award in Environmental Science
• 2005-2006 Environmental Protection Agency Award for Outstanding Project in the
Field of Science
Dale, Z., Thomas, R. Wilson, B. “Characterization of Organic Compounds in the effluent of wastewater
Treatment Plants
• 2008 Historically Black Colleges and Universities Conference (HBCU-UP)
• 2009 Historically Black Colleges and Universities Conference (HBCU-UP) 1st
Place
Prize
• 2009 Southeastern Association of Educational Opportunity Program Personnel
Conference (SAEOPP)3rd
Place Prize
• 2009 13th
Annual Ronald McNair MKN Conference
Dale, Z., Thomas, R Wilson, B. “Assessment of Environmental Estrogens in the Galveston Bay
Watershed.”
• 2010 113th
Texas Academy of Science (TAS) Research Conference

44. 44
• 2010 36th
National Association for the Professional Advancement of Black Chemists and
Chemical Engineers
• AAAS Emerging Researchers National Conference in STEM 2011- 1st
Place Prize
• NOBCChE National Conference in STEM 2011-2nd
Place Prize
Dale, Z., “Clostridium Botulinum”
• 2010 Award for Best Toxic Substance Presentation given to a panel of Centers for
Disease Control and
Prevention (CDC) /Agency for Toxic Substances and Disease Registry (ATSDR)
reviewers
Dale, Z., “Selenium Removal using Biogenic Manganese Oxide”
• NSF/TAMUS/LSAMP 9th
Annual Symposium, 2013
Dale, Zuri, Karthikeyan, R. “Studies on biological removal of Mn (II) in water using Pseudomonas putida
MnB1”
• TWRI/TAMU Water Daze Poster Symposium 04/2013
• Hispanic Leaders in Agriculture and the Environment Annual Research Symposium
2013
245th American Chemical Society (ACS) National Meeting & Exposition, 04/2013-Attendee
EMPLOYMENT/INTERNSHIPS/PROFESSIONAL EXPERIENCES
Texas Parks and Wildlife Intern District 3-E, Snook, Texas, 05/2013-08/2013
• Primarily responsible for identifying stakeholders in the development of San Jacinto
Watershed conservation management plan
• Assisted with fisheries management practices of freshwater community lakes and ponds
• Assisted with fish population monitoring and habitat vegetation planting for Texas
freshwater lakes
• Assisted in analyzing water quality parameters of Biological Oxygen Demand, pH, and
temperature in surface water bodies at project sites
Methods of Technological Change Study Abroad, Trinity and Tobago, West Indies-03/2012
• Reviewed water quality and ecosystem health of Caroni River
• Studied international agricultural technologies and practices.
Texas A&M University, Water Quality Engineering Laboratory, Graduate Student,
08/2011-03/2014
• Assisted with preparing, collecting, and analyzing environmental water samples
• Prepared reports which document laboratory activities
• Performed complex chemical analysis of water samples
• Bacteria Enumeration using standard dilution plating techniques
• Analyzed water samples using colorimetric procedures
STEM READY Internship Program, Texas Southern University 05/2011-08/2011, 05/2012-
08/2012
• Served as Assistant Program Coordinator in the STEM-READY Internship program
whose mission is “to train the next generation of scientists.” Responsibilities included:
• Oversaw and facilitated weekly professional development modules
• Participated in review and evaluation of plans and criteria for projects
• Assisted in planning, organizing, and coordinating activities
• Reported directly to Program Coordinator.
Office of Information Technology, Texas Southern University, 08/2010-05/2011
• Biology tutor.
Centers for Disease Control and Prevention, Chemical Weapons Elimination Branch,
Atlanta, Ga., 06/2010-08/2010

45. 45
• Assisted in drafting a document that detailed the importance of the safe destruction of
the Army’s stockpile of chemical weapons for the protection of public health
• Assisted in Computational Toxicology Project that used QSAR methods to identify non-
carcinogenic effects associated with certain chemicals
• Visited disposal facility locations to ensure compliance with Federal regulations
NASA Students Pursuing Academic and Career Excellence (NSPACE)/Texas Southern
University 09/2008-05/2011
• Worked in a laboratory setting determining the presence of environmental estrogens in
Galveston Bay Watershed performing analysis of water and wastewater samples.
• Assisted with collection and preparation of environmental samples
• Analyzed and interpreted chemical analysis according to established laboratory
procedures
• Analyzed sample components using High Performance Liquid Chromatography,
Inductively Coupled Plasma Mass Spectrometry and Gas Chromatography Mass
spectrometry as well as basic Instrument troubleshooting and maintenance
• Prepared and maintained laboratory supplies to ensure laboratory productivity
• Conducted technical inspections and follow-up investigations at sample collection sites
Mickey Leland Kibbutzim Internship, Tel Aviv, Israel, 06/2006
• Non-profit leadership program that enables students to work, travel and live in Israel for
one month
CLUBS and PROFESSIONAL AFFILIATIONS
American Chemical Society, 05/13-Present
“The Drop” Newsletter Contributing Editor, 08/12-Present
Hispanic Leaders in Agriculture and the Environment Fellow (HLAE) 08/2011-Present
American Water Resources Association (AWRA) 08/2011- Present
Early Medical School Acceptance Program (EMSAP) 06/2010- 06/2011
Student Government Association, Director of Student Initiatives 06/2010-05/2011
Dean Search Committee-04/2010
National Association for the Advancement of Colored People 08/ 2009-05/2010, Co-Chair of
Fundraising
University Program Council Executive Board, 08/2009- May 2010
Ronald E. McNair Scholars Program 05/2009- 05/2011
National Assn. for the Professional Advancement of Black Chemists and Chemical
Engineers 03/2009-Present
Hugh O’Brien Youth Leadership Alumni (HOBY) 06/2005-Present
FELLOWSHIPS & GRANT
Bridge to Doctorate Fellow, Texas A&M University 2011-2013.