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Effect of drinking water treatment processparameters on biological removal of manganesefrom surface water
Victoria W. Hoyland a,*, William R. Knocke a, Joseph O. Falkinham IIIb,Amy Pruden a, Gargi Singh a
a VT Via Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061, USAb VT Biological Sciences Department, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
a r t i c l e i n f o
Article history:
Received 17 May 2014
Received in revised form
30 July 2014
Accepted 5 August 2014
Available online 14 August 2014
Keywords:
Manganese
Biofilters
Manganese-oxidizing bacteria
* Corresponding author. 1901 Innovation DriE-mail address: vlwheaton@gmail.com (V
http://dx.doi.org/10.1016/j.watres.2014.08.0060043-1354/© 2014 Elsevier Ltd. All rights rese
a b s t r a c t
Soluble manganese (Mn) presents a significant treatment challenge to many water utilities,
causing aesthetic and operational concerns.While application of free chlorine to oxidizeMn
prior to filtration can be effective, this is not feasible for surfacewater treatment plants using
ozonation followed by biofiltration because it inhibits biological removal of organics.
Manganese-oxidizing bacteria (MOB) readily oxidize Mn in groundwater treatment applica-
tions, which normally involve pH > 7.0. The purpose of this study was to evaluate the po-
tential for biological Mn removal at the lower pH conditions (6.2e6.3) often employed in
enhanced coagulation to optimize organics removal. Four laboratory-scale biofilters were
operated over apHrange of 6.3e7.3. Thebiofilterswereable to oxidizeMnat apHas lowaspH
6.3 with greater than 98%Mn removal. Removal of simulated organic ozonation by-products
was also greater than 90% in all columns. Stress studies indicated that well-acclimatedMOB
canwithstandvariations inMnconcentration (e.g., 0.1e0.2mg/L), hydraulic loading rate (e.g.,
2e4 gpm/ft2; 1.36 � 10�3e2.72 � 10�3 m/s), and temperature (e.g., 7e22 �C) typically found at
surface water treatment plants at least for relatively short (1e2 days) periods of time.
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Manganese (Mn) isoftenpresent indrinkingwatersources inthe
reduced Mn(II) or oxidized Mn(IV) form. Mn(II) is the more sol-
uble form, and its oxidation results in the formation of a dark
brown MnO2 precipitate, causing unsightly black discoloration
of water and scaling of pipes and fixtures (Sly et al. 1990). Mn in
drinking water can cause aesthetic and operational concerns at
ve, Suite 2100, Blacksburg.W. Hoyland).
rved.
levels below the EPA Secondary Maximum Contaminant Level
(SMCL) of 0.05mg/L (Sly et al. 1990) andhas recently been linked
to neurotoxic effects in children (Wasserman et al. 2006;
Bouchard et al. 2010). One method for soluble Mn removal is to
applyfreechlorineto theinfluentdirectlyupstreamofagranular
filter bed. The result is a natural greensand effect (NGE) where
Mn(II) is initially adsorbed onto a MnOx(s) surface on themedia
and then subsequently oxidized by free chlorine present in the
filter-applied water (Knocke et al. 1988).
, VA 24060, USA.
wat e r r e s e a r c h 6 6 ( 2 0 1 4 ) 3 1e3 932
Removal of organic compounds from drinking water
sources is also a growing concern, especially for surface water
treatment plants. One effective approach is to apply ozonation
in order to enhance the biodegradability of the organic com-
pounds prior to biofiltration (Rittmann et al. 1989). Ozonation
can also result in soluble Mn oxidation, although its effec-
tiveness for Mn removal is often suboptimal (Wilczak et al.
1993).
Application of chlorine across the filter media to achieve
NGE is at odds with biological treatment benefits of the filter.
Ideally, an effective biofiltration strategy that removes both
Mn and organic contaminants is desirable. This could poten-
tially be achieved by employingmanganese-oxidizing bacteria
(MOB), which are able to naturally oxidize Mn(II) to Mn(IV).
Biological removal of Mn has been studied extensively in the
treatment of groundwater and successfully applied at facil-
ities (primarily in Europe) since the 1980s (Mouchet, 1992;
Vandenabeele et al. 1992; Hope and Bott, 2004; Katsoyiannis
and Zouboulis, 2004; Pacini et al. 2005; Stembal et al. 2005; Li
et al. 2006; Burger et al. 2008a; Tekerlekopoulou et al. 2008).
Biological Mn removal has traditionally been thought to be
restricted to pH > 7.0e7.5 (Mouchet, 1992), although biological
Mn removal may be possible at lower pH (6.5) (Burger et al.
2008a, 2008b).
The role of MOB in biofiltration for Mn removal in surface
water treatment situations has been far less researched.
Granger et al. (2014) reported biological Mn removal to be
effective at pH 6.0 with phosphorus enhancement; less
effective Mn removal was noted under elevated pH (9e11)
conditions. Ozonation was not part of the treatment process
stream employed in this study; as such, the organic matter
present in the filter-applied water was not readily biodegrad-
able but, instead, was natural organic matter from a lake
source.
MOB are phylogenetically diverse and include various
species (Tebo et al. 2004) that appear to have evolved the
ability to oxidize Mn independently. Several MOB have been
studied in pure culture, including: Pseudomonas putida strain
GB-1 (Okazaki et al. 1997), Leptothrix discophora strain SS1
(Boogerd and Devrind, 1987; Katsoyiannis and Zouboulis,
2004) and L. discophora strain SP-6 (Hope and Bott, 2004;
Burger et al. 2008b), and Bacillus sp. strain SG-1 (Devrind
et al. 1986; Bargar et al. 2000). In particular, Bacillus sp. strain
SG-1 (Devrind et al. 1986; Bargar et al. 2000) and other Mn-
oxidizing Bacillus (Francis and Tebo, 2002; Cerrato et al. 2010)
are able to oxidize Mn in the dormant spore form. Bacteria in
spore form are known to be resistant to a host of environ-
mental threats, including heat and oxidants (Nicholson et al.
2000).
The overall goal of this study was to evaluate the potential
for simultaneous Mn and organics removal via biofiltration
over a range of pH and loading conditions relevant to surface
water treatment facilities, especially those employing up-
stream coagulation and ozonation. Removal of Mn and or-
ganics was evaluated in four lab-scale anthracite coal
biofiltration columns over a pH range of 6.3e7.3. The resil-
ience of the columns to shifts in Mn concentration, hydraulic
loading rate, and temperature was evaluated. The results of
this study provide insight into the range of conditions
conducive to biological Mn removal in surface water
treatment plants and the corresponding role of microbial
communities in accomplishing Mn removal.
2. Materials and methods
2.1. Laboratory-scale biofilters
Lab-scale glass columns were used to simulate the anthracite
coal layer of a biologically active filter at a surface water
treatment plant. Glass columns with 5 ft (1.52 m) length and
1.5 inch (3.81 cm) inner diameter were constructed with
sampling ports at 4e6 inch (10e15 cm) intervals along the
column length. Anthracite coal media (effective size
0.95e1.05 mm; uniformity coefficient <1.4) were obtained
from a full-scale biofilter at the Newport News Waterworks
Lee Hall Treatment Plant (Newport News, VA). The columns
were inoculated with a mixture of six MOB isolated from four
water treatment facilities and distribution systems (Cerrato
et al. 2010). MnO2(s) deposition capability over a range of pH
(6.0e7.4), rapid growth rate, and representation of available
genera were the primary criteria for isolate selection. The
species classifications of the six MOB isolates were Bacillus
pumilus (MB-2 and MB-3), Lysinbacillus sphaericus (MB-17),
Lysinbacillus fusiformis (MB-22), Pseudomonas aeruginosa (MB-
33), and Brevundimonas nasdae (MB-38). The media were inoc-
ulated with the MOB mixture and distributed into the four
columns to a depth of two feet (0.61m). A six-inch gravel layer
(nominal size of 0.25e0.5 in, 0.64e1.3 cm) supported the
anthracite coal media bed, and the columns were shielded
from light with aluminum foil.
Isolates MB-2, MB-3, MB-17, MB-22, and MB-38 were rein-
oculated after 30 days of operation by recirculating feed water
for four days with 2.5 mg/L Mn(II), as recommended by Hope
and Bott (2004), because only the Pseudomonas isolate MB-33
remained detectable on MOB selective media during start-up.
2.1.1. Feed water systemThe initial feedwater systembefore reinoculation consisted of
tap water from Blacksburg, Christiansburg, VPI Water Au-
thority, dechloraminated using granular activated carbon
(Calgon Centaur 12x40) with an empty bed contact time of
10 min. The average water quality characteristics of the tap
water were as follows: pHe 7.7, alkalinitye 49mg/L as CaCO3,
hardness e 46 mg/L as CaCO3, dissolved organic carbon (DOC)
e 1.2 mg/L, ammonia e 0.73 mg/L, Mn e <0.5 mg/L, nitrate e
0.45mg/L as N, phosphatee 0.24mg/L as P, with N and P levels
sufficient to ensure carbon was the limiting nutrient. The
water temperature wasmaintained above 20 �C and aerated to
achieve 7.5e8.0 ppm dissolved oxygen. The water was pum-
ped into the columns at 2 gpm/ft2 (1.36 � 10�3 m/s) and sup-
plemented with soluble Mn, pH adjustment, and
biodegradable organic matter (BOM). Concentrated sulfuric
acid diluted in distilled water was used for pH adjustment.
Manganous sulfate monohydrate (MnSO4$H2O) was added to
distilled water to achieve a 0.1 mg/L soluble Mn feed to the
columns. The Mn concentration was increased 16 days after
startup to 0.5 mg/L Mn in an attempt to stimulate MOB accli-
mation and was returned to 0.1 mg/L once Mn removal was
established across the filters.
Fig. 1 e Schematic of final filter column setup (after day 50).
wat e r r e s e a r c h 6 6 ( 2 0 1 4 ) 3 1e3 9 33
The BOM added to the feed was intended to simulate post-
ozonation by-products commonly found in water treatment
plants using ozone disinfection. The carbon mixture found in
Elhadi et al. (2006) was modified to target concentrations of
500 mg/L oxalate, 400 mg/L formate, 50 mg/L formaldehyde and
25 mg/L glyoxal. The BOM stock was made by adding sodium
formate, sodium oxalate, 40% glyoxal in water, and 37%
formaldehyde in water with preservatives to high-purity
deionized water from a Nanopure system. The stock
container was shielded from light to minimize degradation of
the carbon compounds.
No Mn removal was observed over the first 30 days of oper-
ation.Microorganisms in theGAC converted the ammonia from
the chloramines into nitrite, so during reinoculation, a break-
point chlorination system was installed prior to the GAC to
oxidize and remove theammoniaasnitrogengas. Thehydraulic
designof thesetupwasalsoreconfiguredduringreinoculationto
relocate BOMaddition directly prior to entry into the columns to
preventBOMdegradationbytubingbiofilm.Fig.1showsthefinal
feedwater system. Further informationanddetails on the initial
feed water system can be found in (Hoyland, 2013).
Table 1 e Summary of laboratory-scale column operating para
Column Initial pH pH after MOB reinoculation Stres
A 6.0 6.3 Mn conc
B 6.3 6.7 None
C 6.7 7.0 Mn conc
Hydrauli
Tempera
D 7.0 7.3 pH
2.1.2. Maintenance and samplingThe columns were backwashed weekly by pumping dechlor-
aminated tap water upflow at approximately 25 gpm/ft2
(1.7 � 10�2 m/s), which corresponded to a 20% bed expansion.
Air in the effluent tube provided an initial air scour before the
backwash water reached the column media. The initial
backwash time was 5 min, which was increased to 7 min
starting Day 93 due to evidence of more extensive head loss in
the columns, most likely from biological growth on the filter
media.
Aqueous samples were withdrawn from sampling ports
using 30 mL syringes with stainless steel needle tips. Influent
samples were collected from the top port above the media
depth and effluent samples were collected from the bottom
sampling port in the gravel layer. Starting on Day 93, samples
for Mn analysis were filtered through 0.45 mm PVDF filters
(Millipore, Billerica, MA) to remove biofilm prior to acidifica-
tion with 2% nitric acid. Media samples were collected by
fluidizing the bed during backwashing, opening the influent
sample port and collecting themedia andwatermixture into a
sterile bottle.
meters.
s test Start day of stress test Duration of stress test
entration 181 24 h
N/A N/A
entration 139 24 h
c loading 157 24 h
ture 226 134 days
233 144 days
wat e r r e s e a r c h 6 6 ( 2 0 1 4 ) 3 1e3 934
2.2. Stress experiments
Table 1 summarizes the conditions of the four columns (AeD)
and influent stress test performed on each column. Aqueous
samples were collected from the influent, effluent, and profile
sample ports for Mn analysis during the stress tests.
2.2.1. Mn increase stress testThe influent Mn concentration was doubled from 0.1 mg/L to
0.2mg/LMn in Columns A (pH 6.3) and C (pH 7.0) for a period of
approximately 24 h by incrementally increasing Mn concen-
tration in the Mn/pH adjustment stock solution over 2 h,
maintaining the higher concentration, and decreasing the Mn
concentration in the same fashion.
2.2.2. Hydraulic loading rate increase stress testInfluent hydraulic loading rate was doubled from 2 gpm/ft2 to
4 gpm/ft2 (1.36 � 10�3e2.72 � 10�3 m/s) in Column C (pH 7.0)
for approximately 24 h. Batched feed water was used during
incremental increasing and decreasing of the hydraulic
loading rate over 2 h. Experimental conditions for Mn con-
centration, filter-applied pH, and BOM concentration were
maintained during the hydraulic loading rate stress study by
adjustments to stock solution concentrations as necessary.
2.2.3. Temperature decrease stress testA chiller was used to decrease the influent water temperature
to Column C (pH 7.0) approximately 0.5 �C per day for 25 days.
Column C was backwashed with chilled dechloraminated tap
water when head loss reached approximately one foot. The
influent temperature was increased over the course of 20 days
once it reached 7 �C. The Mn removal across Column C was
monitored for an additional 30 days after the column returned
to room temperature (22 �C).
2.2.4. Column D pH adjustmentColumn D (pH 7.3) was expected to achieve greater Mn
removal than the other columns because it was operating
closer to the pH range traditionally associated with effective
biological Mn removal (Mouchet, 1992). After 233 days of
operation at pH 7.3, Mn removal in Column D remained sub-
stantially lower than the other lower pH columns. To explore
the effect of pH on Column D performance an additional
“stress” test was carried out in which the influent pH to Col-
umn D was decreased to pH 6.7 starting Day 233 for 60 days,
before returning to pH 7.3.
2.3. Analytical methods
Aqueous samples were preserved in 2% trace metal grade ni-
tric acid for Mn analysis. Samples above 0.05 mg/L Mn were
analyzed using flame atomic absorption spectroscopy (Fl-
AAS) and below 0.05 mg/L using inductively coupled plasma
mass spectroscopy (ICP-MS). The minimum detection limit
(MDL) was 0.017 mg/L for ICP-MS and 13.4 mg/L for Fl-AAS. pH
was measured using an Accumet pH electrode probe and
Oakton pH 110 series meter. Temperature was measured
using a digital Fisher Scientific traceable thermometer.
Formaldehyde and glyoxal were measured following EPA
Method 556 (Munch et al. 1998). The MDLs for formaldehyde
and glyoxal were determined to be 0.64 and 4.5 mg/L, respec-
tively. Formate and oxalate were measured using a modified
version of the ion chromatography method outlined in
Peldszus et al. (1996, 1998). A 70 mM NaOH eluent was used
because equipment limitations prevented using an eluent
gradient. Samples were preserved with 0.1% v/v chloroform
and analyzed immediately after sampling. The MDLs for
formate and oxalate were determined to be 36 and 6.2 mg/L,
respectively. For the purpose of data analysis, all BOM
componentmeasurements below theMDLwere considered to
be half the MDL value.
Mn-oxidation broth and agar plates were prepared ac-
cording to Stein et al. (2001), adjusted to pH 7.0. The supple-
mented reduced Mn present in the medium allows MOB to be
distinguished from other heterotrophs by their characteristic
brown colony color. Incubation temperature was 30 �C. Cellsuspensions were collected by vortexing media samples at
high speed for 1 min. Spores were isolated following Krieg
(1981) and were plated on the Mn-oxidation agar. Repetitive-
sequence-based polymerase chain reaction (rep-PCR)
(Cangelosi et al. 2004) was used to identify which of the five
reinoculatedMOB isolateswere present in the biofilm 200 days
later.
Denaturing gradient gel electrophoresis (DGGE) of bacterial
16S rRNA genes was applied to select media samples (as
described by Singh et al. (2012)) in order to track the fate of the
isolates in the columns and compare the overall microbial
community composition. Amplified 16S rRNA genes from
select samples from Columns B and D were subjected to 454
pyrosequencing (Research and Testing Laboratories, Lubbock,
Texas).
2.4. Data analysis
The relative intensities of visible DGGE bands were trans-
formed by taking the fourth root and Bray Curtis distance and
were used to construct cluster dendrograms using Primer-E
(Clarke and Gorley, 2006). The significance of clustering was
tested using the Simprof test with significance set at p < 0.05.
Pyrosequencing data was analyzed using Mothur v.1.30.0
using 454 SOP (Schloss et al. 2011). Analysis of similarity test
was done using Simprof test from Clustsig package (Oksanen
et al. 2013) in R version 2.15.3 (R Core Team, 2013). Classical
multidimensional scaling (MDS) analysis and plotting of the
graphs was done using packages MASS (Venables and Ripley,
2002), vegan (Oksanen et al. 2013), and rgl (Adler andMurdoch,
2013) in R (R Core Team, 2013).
3. Results and discussion
3.1. Biofilter column acclimation
3.1.1. Effect of influent pH on Mn removalMn removal was observed within one week of Phase 2 startup
(post-reinoculation) in Columns A (pH 6.3) and B (pH 6.7) and
within three weeks in Column C (pH 7.0) (Fig. 2). Essentially
complete (>98%) Mn removal was achieved in Columns A, B,
and C, supporting prior research results which demonstrated
that MOB can oxidize Mn under pH conditions as low as 6.5
Fig. 2 e Influent and effluent Mn concentration during
startup of the lab-scale column (Column A e Phase 1
pH ¼ 6.0, Phase 2 pH ¼ 6.3; Column B e Phase 1 pH ¼ 6.3,
Phase 2 pH ¼ 6.7; Column C e Phase 1 pH ¼ 6.7, Phase 2
pH ¼ 7.0; Column D e Phase 1 pH ¼ 7.0, Phase 2 pH ¼ 7.3).
wat e r r e s e a r c h 6 6 ( 2 0 1 4 ) 3 1e3 9 35
(Burger et al. 2008a). Column D (pH 7.3) did not remove an
appreciable amount of Mn. This was contrary to prior reports
that MOB require pH conditions as high as 7.4e7.5 to oxidize
Mn (Mouchet, 1992). Additionally, the columns operating at
lower pH (6.3 and 6.7) began removingMn earlier than those at
higher pH, supporting the findings of Burger et al. (2008b),
where better Mn removal was observed at pH 6.5 than pH 7.5.
The Burger study was carried out in laboratory-scale filter
studies inoculated with L. discophora strain SP-6 and indige-
nous microflora from full-scale treatment facilities where
biological Mn removal was occurring.
Interestingly, despite negligible Mn removal in Column D
over 200 days of acclimation at pH 7.3, removal was readily
stimulated by decreasing the pH to 6.7 (Fig. 3). Mn removal
reached greater than 98% at pH 6.7, but correspondingly
declined again when the pH was returned to 7.3. It is impor-
tant to note that Mn removal improved markedly (in com-
parison to the initial studies at pH 7.3) when the pH was
adjusted up to 7.3 after operating effectively for several days at
pH 6.7. Although Column D Mn removal decreased to 70%
when the pHwas first increased to 7.3, eventually Mn removal
greater than 98% was achieved. This result may indicate that
once acclimated, the MOB were able to tolerate pH 7.3. Alter-
natively, MOB-based formation of MnOx(s) on the media
Fig. 3 e Effect of influent pH on Mn removal in Column D.
surface at pH 6.7 may have allowed physical-chemical Mn
removal processes to contribute to overall Mn removal. Mn
removal in pilot-scale trickling filters has been attributed to
both biological and physical-chemical mechanisms (Gouzinis
et al. 1998). Most of the Mn removal occurred within the first 4
in. (10.2 cm) of the columns (Fig. 4). This is expected since
most of the biological activity occurs at the top of biofilters,
where concentrations of organic carbon and other nutrients
are highest. Physical-chemical Mn oxidation and removal also
occurs primarily at the top of filter media.
The initial period of poor Mn removal (Column D; pH 7.3)
was unexpected based upon the published work of others
(Mouchet, 1992; Katsoyiannis and Zouboulis, 2004; Li et al.
2006; Burger et al. 2008a; Katsoyiannis et al. 2008). A defini-
tive explanation for this difference in performance was not
achieved in the current study. All of the research work cited
above involved the treatment of groundwater sources which
would typically be characterized by low DOC concentrations,
with minimal BDOC present. In contrast, the waters in the
current study had a significant amount of BDOC present.
Future research could investigate whether the presence or
absence of BDOC may impact MOB effectiveness for soluble
Mn removal under alkaline (>pH 7) conditions.
3.1.2. Acclimation timeMn removal was not observed in any of the columns over the
first 50 days (Phase 1) at which time several factors were
adjusted simultaneously in an attempt to stimulate removal:
a) columns were reinoculated with MOB; b) the chloramine
removal method was changed, which changed the form of
nitrogen composition of the influent water; c) Mn influent
concentration was increased from 0.1 mg/L to 0.5 mg/L; and d)
the hydraulic setup was changed to prevent BOM degradation
prior to the columns and provide consistent influent flow.
Because multiple factors were changed simultaneously, it is
not possible to concludewhich, if any, contributed to the rapid
onset of Mn removal in Columns AeC from Day 50 onwards
(Phase 2).
Regardless, acclimation appears to be an important factor
for MOB. Even upon the onset of Mn removal, nearly complete
(98%) removal was not achieved for an additional 50 days.
Fig. 4 e Mn removal profiles across the media depth of the
lab-scale columns once 98% Mn removal was established
in Columns A, B, and C. Profile samples collected on Day
220.
Fig. 5 e Mn removal profile across Column C filter media
depth during influent Mn concentration increase stress
study (influent pH of 7.0).
Fig. 6 e Effect of temperature on Mn removal in Column C
(influent pH of 7.0). Time is displayed as days since
temperature study began. (Day 0 is 226 days after Phase 1
startup).
wat e r r e s e a r c h 6 6 ( 2 0 1 4 ) 3 1e3 936
Mouchet (1992) documented a similar slow improvement in
Mn removal with longer acclimation times, with the time
between initial Mn removal and near complete removal
requiring approximately 35 days. Additionally, Burger et al.
(2008b) found that a lab-scale groundwater MOB biofilter
required approximately 45 days for acclimation.
The increased Mn concentration during Phase 2 startup of
the lab-scale columns is one factor that may have aided the
eventual stimulation of Mn removal. As previously
mentioned, most studies on the use of MOB in biofiltration
have involved groundwater systems which generally have
higher influent Mn concentration than surface water treat-
ment plants (Mouchet, 1992; Vandenabeele et al. 1992; Hope
and Bott, 2004; Katsoyiannis and Zouboulis, 2004; Li et al.
2006; Burger et al. 2008a). Hope and Bott (2004) recom-
mended an initial Mn concentration of 2.5 mg/L in a recircu-
lation setup for fastest acclimation. The lab-scale columns
were operated in this configuration during reinoculation in
Phase 2 for the first week of operation before starting normal
flow at 0.5 mg/L Mn. This may have contributed to the Mn
removal increase shortly after Phase 2 reinoculation.
3.1.3. Biodegradable organic matter removalBOMwas readily removed across all columns, ranging from 90
to 98% removal for the four compounds (formate, oxalate,
glyoxal, formaldehyde). As BOM removal is the primary pur-
pose of biofiltration, it was an important finding that MOB
inoculation did not adversely affect this process. Similar to
Mn, most (>80%) of the BOM removal occurred in the first 4 in.
(10.2 cm) of the column, as shown in Fig. S-1 in the supple-
mental information.
3.2. Stress experiments
3.2.1. Mn concentrationEffluent Mn concentration did not appreciably increase when
influent Mn concentration doubled from 0.1 to 0.2 mg/L over a
24 h period in Column C (pH 7.0), indicating that acclimated
MOB communities are resilient to short-term variations in Mn
concentration. Influent shifts are common in surface water
treatment plants due to changes in upstream treatment pro-
cesses. Burger et al. (2008b) measured similar results in
acclimated lab-scale groundwater MOB biofilters. The Mn
removal profiles did shift slightly towards higher Mn levels
persisting further down the media depth (Fig. 5), indicating
there may be long-term effects on Mn removal if elevated Mn
levels had persisted. Seasonal long-term variations in Mn
concentration are common at surface water treatment plants;
however, these changes are typically gradual and can be
addressed by changes in upstream treatment processes and/
or MOB acclimation to the higher influent Mn level. Similar
results were observed in aMn increase stress study in Column
A conducted at pH 6.3 (see Fig. S-2).
3.2.2. Hydraulic loading rateHydraulic loading rate may also vary to some degree with
demand in drinking water treatment plants. The results of the
hydraulic loading rate increase stress study of Column C (pH
7.0) were similar to that of the Mn increase stress study. Mn
breakthrough to the filter effluent did not occur during the 24-
h stress period, but the Mn removal profile did shift slightly
over time (Fig. S-3). Stembal et al. (2005) found similar shifting
of Mn removal profiles in groundwater biofilters at increasing
hydraulic loading rates; however, effluent Mn concentration
remained unchanged when the hydraulic loading rate was
doubled.
3.2.3. TemperatureThe lab-scale column temperature decrease study indicated
that well-acclimated MOB can withstand temperatures com-
mon in a mild winter condition (Fig. 6). Mn removal decreased
as influent temperature decreased in Column C (pH 7.0) and
increased once the water temperature began to increase,
eventually returning to greater than 98% Mn removal. Soluble
Mn in surface water sources is typically a summer and fall
concern, when water temperatures would normally be
warmer. As such, biological Mn removal at colder tempera-
tures is not generally necessary, but the ability for MOB to
survive winter conditions on the filter media and readily
removeMn once temperatures increase is critical for full-scale
applications. These results indicate the MOB were not only
able to survive and recover from colder winter temperatures,
they were also able to maintain some oxidative activity
throughout the colder temperature conditions.
Fig. 7 e A) Cluster analysis of denaturing gradient gel
electrophoresis (DGGE) profiles of bacterials 16S rRNA
genes in the four columns. Analysis of D columns is before
and after experiment testing effect of temporarily lowering
the pH. Black lines separate significantly different clusters.
B) Multi-dimensional scaling analysis of pyrosequencing of
bacterial 16S rRNA genes in Column B versus Column D,
before and after experiment testing effect of temporarily
lowering the pH.
wat e r r e s e a r c h 6 6 ( 2 0 1 4 ) 3 1e3 9 37
3.3. Microbial composition
MOB have been reported to comprise a large percentage
(25e33%) of the total microbial population in mature
groundwater treatment biofilters (Vandenabeele et al. 1992).
The percentage of MOB in mature surface water treatment
biofilters may be much smaller. The percentage MOB with
respect to total heterotrophic plate count (HPC) in the lab-
scale columns was determined using agar plating methods
on Days 104 and 118 when Columns B (pH 6.7) and C (pH 7.0)
were removing greater than 98%Mn, Column Awas removing
around 70% Mn, and Column D was not appreciably removing
Mn. MOB accounted for a very small percentage of total HPC
(3.6% in Column A, 5.7% in Column B, 1.3% in Column C, and
2.1% in Column D); however, all isolated spore-forming col-
onies from each column were identified as MOB.
The rep-PCR analysis indicated that the majority of the
MOB colonies and spores present in the media biofilm corre-
sponded to B. pumilus, which corresponded to strain MB-3
originally isolated by Cerrato et al. (2010) from the Newport
News Lee Hall water treatment plant where the lab-scale
media was obtained (see Figs. S-4 and S-5 and Table S-1).
Bacilli may require stress to induce Mn oxidation as these
species are known to oxidize Mn only in their spore form
(Devrind et al. 1986; Bargar et al. 2000), which could also be a
reason for the extended acclimation time. The MB-3 growth
curves (see Fig. S-6) may provide further insight as to why
ColumnDwas unable to initially remove substantial Mn at pH
7.3. The lag phase of MB-3 is much longer at pH 7.4 than at pH
6.0 or 6.5. MB-3 may grow and oxidize Mn differently at higher
pH than lower pH.
The DGGE cluster analysis results indicated that the mi-
crobial communities present on Column D media before the
pH change (Day 183, 20%Mn removal) and after the pH change
(Day 260, 65% Mn removal) were not statistically different
(p < 0.05, Simprof test). Further, both Column D microbial
communities were different from samples collected (on Day
183) from Columns A, B, and C (p < 0.05 Simprof test.), which
were not significantly different from each other (p < 0.05
Simprof test.). These results suggest that the higher pH in
Column D selected for a distinct microbial community and
that the microbial community directly impacts Mn removal.
Notably, the microbes in Column D exhibited distinct Mn
removal behavior relative to the other columns. Nonetheless,
they did appear capable of acclimating when the pH condi-
tions were temporarily decreased. Further information on the
DGGE results can be found in Tables S-2 and S-3 and Fig. S-7.
Pyrosequencing was applied to compare the bacterial com-
munities in Columns B and D, which exhibited contrasting
performance and harbored distinct bacterial communities
based onDGGE results. Pyrosequencing results suggest that the
bacterial community inColumnDclustered separatelyandwas
significantly different from the bacterial community in column
B (p<0.05, Simprof test)during theiroperation inPhase2 (Fig. 7).
Further, the bacterial community composition of ColumnDdid
not shift significantly during the course of the pH change and
subsequent improvedMn removal from ColumnD (Fig. 7). This
suggests that improvedMn removal in ColumnD following the
pHadjustment to 6.7 and return topH7.3wasnot the result of a
shifting bacterial community but, rather, acclimation of the
existing MOB, such as triggering of spore formation.
4. Conclusion
Laboratory-scale studies demonstrated that MOB have po-
tential for soluble Mn control in surface water treatment
plants. Insight into the ability of MOB to oxidize Mn during
surface water treatment plant over a range of conditions has
been gained. The following conclusions can be drawn from
the results of these experiments:
� MOB can oxidize and achieve >98% removal of soluble Mn
in a biofilter as low as pH 6.3, at a condition which surface
water treatment plants often operate.
� Biological Mn removal can be achieved without adversely
affecting BOM removal (>90% removal of BOM compo-
nents), allowing biofilters to achieve their primary
objective.
wat e r r e s e a r c h 6 6 ( 2 0 1 4 ) 3 1e3 938
� Once acclimated, MOB can tolerate short-term (e.g. 1e2
days) changes in influent characteristics commonly
occurring at surface water treatment plants, including
influent increases in Mn concentration (from 0.1 to 0.2 mg/
L) and hydraulic loading rate (from 2 gpm/ft2 to 4 gpm/ft2
(1.36 � 10�3e2.72 � 10�3 m/s)) without Mn breakthrough
occurring (>98% Mn removal). Long-term effects of these
changes may be more pronounced.
� MOB can withstand and recover from an influent water
temperature decrease from 22 �C to 7 �C, simulating a mild
winter.
� Pyrosequencing and DGGE indicated that the bacterial
community in Column D was significantly different from
those of Columns A, B and C. This difference in bacterial
community in Column Dmay have contributed to the poor
Mn removal noted initially at pH 7.3. Also, the pH adjust-
ment between 6.7 and 7.3 for Column D did not result in a
significant shift in bacterial community. This suggests that
different bacterial communities may display differing Mn
removal behavior with varying treatment conditions.
� Overall, acclimation and stress events may be more
important factors for successfully achieving Mn removal
by microbial means than other studies on the application
of biological Mn removal in drinking water treatment have
indicated.
Acknowledgments
The authors would like to thank the Via Department of Civil
and Environmental Engineering and the Biological Sciences
Department at Virginia Tech for providing support for this
work.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.08.006.
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