Science of the Total Environment 347 (2005) 35 – 45

www.elsevier.com/locate/scitotenv

Use of starch and potato peel waste for perchlorate

bioreduction in water
Benedict C. Okeke, William T. Frankenberger Jr.*
Department of Environmental Sciences, University of California, Riverside, CA 92521, USA
Received 12 June 2004; received in revised form 6 November 2004; accepted 9 December 2004
Available online 7 March 2005

Abstract

The cost of carbon substrates for microbial reduction of perchlorate (ClO4 ) is central to the success and competitiveness of a
sustainable bioremediation strategy for ClO4 . This study explored the potential application of starch in combination with an
amylolytic bacterial consortia and potato peel waste for ClO4 bioreduction. We obtained a potent amylolytic bacterial
consortium that consisted of a Citrobacter sp. S4, Streptomyces sp. S2, Flavobacterium sp. S6, Pseudoxanthomonas sp. S5,
Streptomyces sp. S7, and an Aeromonas sp. S8 identified by 16S rDNA sequencing. ClO4 concentration substantially
decreased in purified starch medium inoculated with the amylolytic bacterial consortium and Dechlorosoma sp. perclace. Potato
peel waste supported ClO4 reduction by perclace with the rate of ClO4 reduction being dependent on the amount of potato
peels. Over 90% ClO4 removal was achieved in 4 days in a single time point experiment with 2% (w/v) potato peels waste.
ClO4 reduction in a non-sterile 0.5% potato peel media inoculated with perclace occurred with an initial concentration of
10.14F0.04 mg L 1 to 2.87F0.4 mg L 1 (71.7% reduction) within 5 days. ClO4 was not detected in the cultures in 6 days. In a
non-sterile 0.5% potato media without perclace, ClO4 depletion occurred slowly from an initial value of 9.99F0.15 mg L 1 to
6.33F0.43 mg L 1 (36.63% reduction) in 5 days. Thereafter, ClO4 was rapidly degraded achieving 77.1% reduction in 7 days
and not detected in 9 days. No susbstantial reduction of ClO4 was observed in the sterile potato peel media without perclace in
7 days. Redox potential of the potato peel cultures was favorable for ClO4 reduction, decreasing to as low as 294 mV in 24 h.
Sugar levels remained very low in cultures effectively reducing ClO4 and was substantially higher in sterilized controls. Our
results indicate that potato peel waste in combination with amylolytic microorganisms and Dechlorosoma sp. perclace can be
economically used to achieve complete ClO4 removal from water.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Perchlorate; Water; Bioremediation; Carbon substrates; Starch hydrolysis; Potato peel waste

1. Introduction
* Corresponding author. Tel.: +1 909 787 3405; fax: +1 909 787
2954.
Perchlorate (ClO4 ) is detected in ground water
E-mail address: william.frankenberger@ucr.edu throughout the United States, because ammonium
(W.T. Frankenberger). ClO4 was extensively used in solid propellants for
0048-9697/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2004.12.041
36 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45
rockets, missiles, explosives, and pyrothechnics
(Urbansky, 1998; Gullick et al., 2001; Logan,
2001; Xu et al., 2003). ClO4 salts are highly soluble
in water (2000 g L 1 for sodium perchlorate) and
adsorbs poorly in soil (Xu et al., 2003). Conse-
quently, natural water sources have been contami-
nated with ClO4 . ClO4 is recalcitrant in the
environment and is potentially toxic to various forms
of life (Lamm et al., 1999) primarily due to adverse
effects on the thyroid gland (Capen, 1994; Von Burg,
1995). Recent studies revealed that low concentra-
tions of ClO4 inhibit iodide uptake in humans as
well as animals (Lawrence et al., 2000; U.S. EPA,
2002). ClO4 contamination of wildlife and vegeta-
tion can have adverse effects on the growth of
amphibians (Goleman et al., 2002). The potential
impact of ClO4 on human health has spurred
regulatory agencies to regulate ClO4 concentrations
in drinking water. In California, the Department of
Health Services (CDHS) established an action level
of 6 Ag L 1 for potable water. The CDHS found
ClO4 in drinking water wells in Riverside County
up to 29 Ag L 1 and up to 325 Ag L 1 in San
Bernardino County. High concentrations of up to
180,000 Ag L 1 in ground water not associated with
contamination of public drinking water supplies
were, however, found in Santa Clara County (CDHS,
2004a,b).
Remedial strategies for removal of ClO4 in
ground water, include membrane, ion-exchange
(IX), and biological technologies. Physicochemical
water treatment technologies are expensive and less
attractive for ClO4 removal from ground water.
Microbial reduction of ClO4 to environmentally-
acceptable innocuous end products is currently an
area of intense interest because this technology is
relatively cost-effective and environmentally compat-
ible. Although several studies have addressed bio-
remediation of ClO4 (Romanenko et al., 1976;
Wallace et al., 1996; Logan, 1998; Herman and
Frankenberger, 1998, 1999; Urbansky and Schock,
1999; Logan et al., 2000; Miller and Logan, 2002;
Giblin et al., 2000a,b; Frankenberger and Herman,
2002; Giblin et al., 2002; Giblin and Frankenberger,
2001; Losi et al., 2002; Logan et al., 2001; Okeke et
al., 2002; Bender et al., 2002; Okeke and Frank-
enberger, 2003), the cost of substrates will be a
major contributor to the economics of bioremediation
of ClO4 . Organic and inorganic electron donors
have been considered for ClO4 reduction in auto-
trophic and heterotrophic systems, respectively (Xu
et al., 2003). Organic carbon substances used as
electron donors include: acetate (Herman and Frank-
enberger, 1999; Kim and Logan, 2001), acetic acid
(Togna et al., 2001), ethanol (Hatzinger et al., 2000;
Greene and Pitre, 2000; Togna et al., 2001),
methanol (Hatzinger et al., 2000; Greene and Pitre,
2000), lactate (Brown et al., 2000), pyruvate (Brown
et al., 2000), protein nutrients such as brewers yeast,
cottonseed protein or whey (Attaway and Smith,
1993), and molasses (Okeke et al., 2002). Hydrogen
has been used in autotrophic systems for ClO4
reduction (Giblin et al., 2000a,b; Miller and Logan,
2000; Nerenberg et al., 2002).
There are many agro-industrial processes that
generate wastes that can be used as substrates for
microbial growth (Mahmood et al., 1998). Micro-
organisms are capable of utilizing various compo-
nents of the organic matter in such wastes as a
carbon source for growth and for synthesis of
cellular biomass as well. The most abundant solid
component of plant materials is carbohydrates. The
carbohydrate fraction of plants includes mono- and
disaccharide sugars, starch, and structural compo-
nents such as cellulose, lignin, proteins, pectic
substances, and hemicelluloses which are closely
related. Starch is the principal reserve polysaccharide
found in many economically important crops such as
potato, wheat, rice, maize, and tapioca (Nirmala and
Muralikrishna, 2003; Van der Maarel et al., 2002).
Large-scale industrial starch processing has emerged
in the last century (Van der Maarel et al., 2002). A
significant portion (30–70%) of the dry weight of
these wastes is carbohydrates other than cellulose
and is therefore readily assimilatable by micro-
organisms (Forage, 1979). Potato peel waste was
chosen for this study. Mahmood et al. (1998)
demonstrated that the solid matter of the potato
peels is predominantly starch. In processing, 20–
59% of the raw product can be generated as waste
(Sistrunk et al., 1979). It is thus a suitable source of
simple and complex carbohydrates for ClO4 reduc-
tion. This study explores the potential application of
starch in combination with an amylolytic bacterial
consortium and potato peel waste for ClO4 reduc-
tion in water.
 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45
2. Materials and methods
2.1. Microorganisms
The efficient ClO4 reducing bacterium, perclace
(Herman and Frankenberger, 1999) identified as a
Dechlorosoma sp. perclace (Xu et al., 2003) was used
in this study. Amylolytic bacterial consortia were
enriched from a mixture of soils and decaying leaf
litter. The sample was pulverized using a mortar and
pestle. Five grams of the sample was inoculated into
triplicate 100 mL sterile FTW mineral salts solution,
to which soluble starch (1 g/L) was added. FTW
medium (Herman and Frankenberger, 1999) is com-
prised (in g/L) of the following: K2HPO4, 0.225;
KH2PO4, 0.225; (NH4)2SO4, 0.225; MgSO47H2O,
0.05; CaCO3, 0.005; FeCl2.4H2O, 0.005, and 1 mL of
trace elements solution (Focht, 1994). The medium
was sterilized by autoclaving (121 8C, 15 min) before
adding the soil sample. The pH of the medium was
adjusted to 7.4 before autoclaving (pH 7.2 after
autoclaving). The aerobic cultures were incubated at
ambient temperature (22 8C) with orbital shaking (180
rpm) for 1 week (round 1 enrichment culture).
Thereafter, 1 mL of the culture was transferred onto
a fresh sterile FTW enrichment medium plus 1 g/L
starch; and further incubated for 1 week (round 2
enrichment culture).
2.2. Characterization of the amylolytic bacterial
enrichment culture
The triplicate cultures were pooled together.
Bacterial composition of the amylolytic bacterial
enrichment cultures was purified by plating on the
enrichment medium solidified with 2% agar. Plates
were incubated aerobically at 25 8C. Colonies were
isolated, purified by repeated streaking, and main-
tained on the same agar medium at 4 8C. Amylolytic
bacterial isolates were identified by 16S rDNA
sequencing as follows: DNA was extracted from the
suspension according to the method described by
Ausubel et al. (1987). Briefly, five to ten colonies of
each bacterial isolate suspended in a mixture of TE
buffer, SDS, and proteinase K, were incubated for 1 h
at 37 8C. NaCl (5 M) and CTAB/NaCl solution (4.1 g
NaCl and 10 g CTAB [N-cetyl-N,N,N,-trimethylam-
moniumbromide] in pre-warmed 100 mL distilled
37
water) were added and incubated for 10 min at 65 8C.
The solution was extracted by adding 780 AL of
chloroform-isoamyl alcohol (24:1), and centrifuged
for 5 min to separate aqueous and organic phases. The
aqueous phase containing the DNA was further
extracted with an equal volume of phenol-chloro-
form-isoamyl alcohol (25:24:1) to remove impurities.
DNA present in the aqueous phase was precipitated
with 0.6 volumes of isopropanol and the DNA
precipitate was washed with 70% (v/v) ethanol. The
DNA pellet was dried using a lyophilizer and re-
suspended in nuclease-free water. Universal bacterial
primers corresponding to E. coli positions 27F and
519R were used for PCR amplification of 16S rRNA
gene. PCR master mix (Cat. No. M7502, Promega,
Madison, WI) was used according to the manufactur-
er’s instructions. Genomic DNA (1 AL) was the
template. DNA was amplified using a 35-cycle PCR
(initial denaturation, 95 8C for 5 min; subsequent
denaturation, 95 8C for 1 min; annealing temperature,
48 8C for 1 min; extension temperature was 728C for
1 min; and final extension was 728C for 5 min). The
PCR product was analyzed on 2% agarose gel to
visualize the product and was purified using a
Microcon YM-10 centrifugal filter (Millipore Corpo-
ration, Bedford, MA). DNA cycle sequencing was
done with the ABI Prism BigDye terminator kit
(Perkin Elmer Applied Biosystems, Foster City, CA)
and an Applied Biosystems ABI 3100 genetic
analyzer.
2.3. Carbon substrates
Soluble starch powder (J.T. Baker, Philipsburg, NJ)
and potato peels were used. Potato peels (white and
red skin) were air dried at room temperature (22 8C)
for 2 days. The potato peels were chopped into small
pieces (approximately 1 cm1 cm0.1 cm). Moisture
content of the potato peels was estimated after drying
at 65 8C for 24 h to be 17.0F0.05% and 27.92F1.0%
for the white and red peels, respectively.
2.4. ClO4 removal by the starch-degrading consortia
and Dechlorosoma sp. perclace
The medium was comprised of 150 mL of FTW
mineral solution (pH 7.4) in 250 mL Erlenmeyer
flasks plus either starch (1% w/v) or potato peels (2%
38 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45
w/v). In independent parallel experiments, 350 AL of
amylolytic bacterial consortia (OD600 of one-tenth
dilution=0.576) and/or the ClO4 -respiring bacterium,
Dechlorosoma sp., perclace (OD600 of one-tenth
dilution=0.538) were added. Cultures were incubated
at room temperature (approximately 22 8C). The white
potato peel was selected for further studies and the
effects of different concentrations were tested in the
range of 0.5–2%, w/v.
2.5. Time course of ClO4 removal using potato peels
as carbon source
This was conducted in 250 mL Erlenmeyer flasks
containing 150 mL of FTW mineral solution and
0.5% (w/v) potato peels. One set was sterilized by
autoclaving (121 8C for 15 min) and the other was
non-sterile. Both sterile and non-sterile medium were
each inoculated with 180 AL of Dechlorosoma sp.
perclace (OD600 of one-tenth dilution=0.221). Flasks
were stoppered with rubber stoppers and cultures
were then incubated at room temperature (22 8C).
Aliquots of the cultures were removed daily for
analysis.
2.6. Redox potential (Eh) and pH
A model 720A pH/ISE meter (Thermo Orion,
Beverly, MA) was used to measure pH and redox
potential (Eh). Eh was measured with an Accumet
combination platinum electrode (Ag/AgCl). The Eh
electrode was tested by immersion in pH 4 and 7 buffer
solutions saturated with quinhydrone (Q2H2) before
each analysis. The measured potential (Ehmeasured) was
converted to actual Eh of starch and potato peels
solution (Ehactual) relative to a standard H electrode as
follows: Ehactual=Ehmeasured+224.4 mV (Jayaweera
and Biggar, 1996).
2.7. Determination of sugar content of cultures
This was determined by the dinitrosalicyclic acid
(DNS) method (Miller, 1959). Briefly, 0.5 mL sample
and 0.5 mL DNS were mixed and the mixture was
heated in boiling water for 10 min to develop the red–
brown color. Thereafter 1 mL of 40% potassium–
sodium-tartarate was added and then cooled to room
temperature. Absorbance was read at 575 nm and
sugar was calculated from a glucose standard curve
( Y=0.336X+0.0016; r 2=0.999).
2.8. Protein determination
Protein was quantified by the standard Bradford
method (Bradford, 1976) using CoomassieR protein
assay reagent (Pierce, Rockford, IL). Briefly, 50 AL of
supernatant sample was added to 1 mL CoomassieR
reagent. Absorbance was read at 595 nm using a
Varian Cary 1E UV–Visible spectrophotometer.
Protein concentration was then quantified from the
following regression equation: Y=0.0031X+0.0058,
r 2=0.997.
2.9. Analysis of ClO4 and other anions
High concentrations of ClO4 (1 to 10 mg L 1)
were analyzed using a ClO4 specific electrode (model
93–81, Orion Research, Boston, MA). Lower con-
centrations (b1 to 0.004 mg/L) were determined by
ion-chromatography. Samples were filtered using a
membrane filter (0.22 Am). ClO4 concentration in the
filtrate was analyzed using a Dionex ion-chromatog-
raphy system (Dionex, Sunnyvale, CA), equipped
with a GP40 gradient pump, an AS40 automated
sampler, 740 AL injection loop, an ionPac AS11
column, and an ED40 conductivity detector. The
eluent was 100 mM NaOH at 1 mL min 1. An ASRS-
II (4 mm) suppressor, operated by external regener-
ation using 50 mM H2SO4 to suppress the eluent was
used.
3. Results
3.1. Starch degrading bacterial consortia
By using starch as the sole source of carbon and
energy, an efficient starch degrading bacterial con-
sortium was achieved after three rounds of enrichment.
Reaction of an aliquot of the enrichment culture with
iodine produced no blue black color indicating
complete breakdown of starch to low molecular weight
compounds. Six bacteria capable of growing on starch
as the sole source of carbon and energy were isolated
from the amylolytic bacterial enrichment. The isolates
were identified by analysis of 16S rDNA as Citro-
 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45 39

Table 1 3 and 4). In the three treatments presented in Table 3,

Bacterial composition of the amylolytic consortia characterized by substantial removal of ClO4 was achieved with the
16S rDNA sequence
white potato peels. In the medium inoculated with
Isolate code Identity Accession Similarity
amylolytic bacteria, amylolytic bacteria plus Dichlor-
number (%)
osoma sp. perclace, and singly with Dichlorosoma sp.
IsoS2 Streptomyces sp. AY148077 98
perclace: 70.2, 90.8, and 93.4% removal of ClO4
IsoS4 Citrobacter sp. AF025370 97
IsoS5 Psedoxanthomonas sp. AY124375 97 were observed, respectively. Less reduction was
IsoS6 Flavobacterium sp. AB075232 98 achieved with red potato peels. With this carbon
IsoS7 Streptomyces sp. AY079156 98 source, 56, 81.4, and 85.3% removal of ClO4 were
IsoS8 Aeromonas sp. AY178568 97 observed in the media inoculated with amylolytic
bacteria, amylolytic bacteria plus Dichlorosoma sp.
bacter sp. S4, Streptomyces sp. S2, Flavobacterium perclace, plus singly with Dichlorosoma sp. perclace,
sp. S6, Pseudoxanthomonas sp. S5, Streptomyces sp. respectively. Redox potential decreased to less than
S7, and Aeromonas sp. S8, respectively (Table 1). 226 in 4 days. Reductions in culture pH were
generally observed to occur in both cultures contain-
3.2. ClO4 removal using purified starch as a carbon ing the white and red potato peels (Tables 2 and 3).
source ClO4 removal was found to be directly related to the
amount of potato peels added (Fig. 1). ClO4 removal
Table 2 presents data on ClO4 removal from water was highest in a 2% potato peel culture. However,
using starch as the sole source of carbon and energy. extended incubation (7 days) revealed that the
In media containing: starch plus the amylolytic addition of 0.5% potato peel was sufficient for
bacterial consortium, and starch plus Dechlorosoma complete removal of ClO4 .
sp. perclace, no perchlortae reduction was observed in
4 days. ClO4 concentration substantially decreased in 3.4. Time course of ClO4 reduction
the starch medium inoculated with the amylolytic
bacterial consortium plus Dechlorosoma sp. perclace Fig. 2 presents the time course of ClO4 removal
achieving over 30% reduction in 4 days. Redox from water using potato peels as the sole carbon
potential decreased substantially in the three media source in non-sterile and sterile media inoculated with
to about 207F5 in 4 days. Significant decreases in Dichlorosoma sp. perclace. ClO4 reduction in the
pH, were recorded in the starch medium inoculated non-sterile potato peel media inoculated with Dichlor-
with the amylolytic bacteria, but no substantial change osoma sp. perclace occurred with an initial concen-
in the pH, was noted with inoculation with perclace. tration of 10.14F0.04 mg L 1 to 2.87F0.4 mg L 1
(71.7% reduction) within 5 days. ClO4 was not
3.3. ClO4 removal using potato peel waste as a detected in the culture in 6 days. In the non-sterile
carbon source potato media that had not been inoculated with
Dichlorosoma sp. perclace, ClO4 depleted slowly
White and red potato peels were evaluated as from an initial value of 9.99F0.15 mg L 1 to
carbon sources for ClO4 removal from water (Tables 6.33F0.43 mg L 1 (36.63% reduction) in 5 days.

Table 2
Effect of inoculation of the amylolytic bacterial consortia and Dechlorosoma sp. perc1ace on ClO4 removal using soluble starch (1%, w/v) as
carbon source
Treatments ClO4 (mg/L) Redox potential (Eh) pH
Initial Final Initial Final Initial Final
Starch+amylolytic bacteria 10.22F0.12 10.14F0.17 376F3 208F7 7.22F0.04 5.03F0.11
Starch+perclace 10.32F0.15 10.25F0.21 381F5 209F6 7.22F0.01 7.03F0.03
Starch+amylolytic bacteria+perclace 10.36F0.28 7.15F0.11 379F5 207F5 7.24F004 5.10F0.04
Cultures were incubated at 22 8C for 4 days. Results are means of triplicate independent experimentsFstandard deviation (N 1).
40 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45

Table 3
ClO4 removal using white potato peels (2%, w/v) as a carbon source with the amylolytic bacterial consortia and Dechlorosoma sp. perclace
Treatments ClO4 (mg/L) Redox potential (Eh) pH
Initial Final Initial Final Initial Final
Potato peels+amylolytic bacteria 10.20F0.29 3.04F0.08 387F4 278F10 7.22F0.03 5.82F0.02
Potato peels+perclace 10.12F0.15 0.67F0.09 382F7 284F1.1 7.28F0.03 5.79F0.12
Potato peels+amylolytic bacteria+perclace 10.25F0.51 0.94F0.08 392F4 279F13 7.23F.01 5.81F0.03
Cultures were incubated at 22 8C for 4 days. Results are means of triplicate independent experimentsFstandard deviation (N 1).

Thereafter, ClO4 was rapidly degraded achieving
77.1% reduction in 7 days. IC analysis of this medium
after day 9 revealed no detectable level of ClO4 . In a
sterile potato peel medium inoculated with Dichlor-
osoma sp. perclace, ClO4 decreased slowly from
10.08F0.02 mg L 1 to 8.47F0.23 mg L 1 (15.97%
reduction) in 5 days and to 6.63F0.49 mg L 1
(34.23%) in 7 days. No susbstantial reduction of
ClO4 was observed in the sterile potato peel medium
that had not been inoculated with Dichlorosoma sp.
perclace during a 7 day incubation period.
3.5. Sugar levels in potato peel cultures
Sugar content of the potato peel cultures was
estimated by the DNS method and is presented in Fig.
3. In general, the concentration of sugar was highest
in the sterile potato peel media. Sugar levels in both
the inoculated and uninoculated non-sterile potato
peel media decreased substantially after 1 day and
thereafter remained very low with growth of the
culture during the 7 days of incubation. In the sterile
potato peel medium inoculated with Dichlorosoma sp.
perclace, the sugar level decreased substantially from
0.14 mg L 1 to 0.09 mg L 1 in 2 days. Analysis of
the sterile potato media that had not been inoculated
with Dichlorosoma sp. perclace showed a sharp rise
in sugar level from day 2 (0.15 mg L 1) to day 4 (0.51
mg L 1) and thereafter stabilized.
3.6. Changes in redox potential
Changes in redox potential of the potato peel media
are presented in Fig. 4. The redox potential dropped
rapidly reaching 291.8 mV, 293.5 mV, and 128
mV in the non-sterile medium inoculated with
Dichlorosoma sp. perclace, the non-sterile medium
without inoculation, and the sterile medium inoculated
with Dichlorosoma sp. perclace, respectively, in 24 h.
Redox readings remained low in these three cultures
between day 1 and day 4. Thereafter, a sharp increase
in redox potential was recorded in the non-sterile
media from day 4 to day 5 and was subsequently
stable at about 100 mV from day 6 to day 7. Redox
potential increased only slightly from day 4 in the
sterile potato peel medium inoculated with Dichlor-
osoma sp. perclace. In the uninoculated sterile potato
peel medium, redox potential decreased only slightly
reaching only 47.43 mV in 4 days and subsequently
stabilized.
3.7. Soluble protein in cultures
Changes in soluble protein content were used to
monitor microbial growth in the potato peel media
(Fig. 5). In the non-sterile potato peel media
inoculated with Dichlorosoma sp. perclace and the
non-inoculated medium, protein levels generally
increased. A sharp increase was observed in the first

Table 4
ClO4 removal using red potato peels (2%, w/v) as carbon source with the amylolytic bacterial consortia and Dechlorosoma sp. perclace
Treatments ClO4 (mg/L) Redox potential (Eh) pH
Initial Final Initial Final Initial Final
Potato peels+amylolytic bacteria 9.74F0.11 4.29F0.23 397F3 235F8 7.22F0.02 5.78F0.04
Potato peels+perclace 9.95F0.68 1.46F0.35 407F3 226F21 7.22F0.02 5.80F0.14
Potato peels+amylolytic bacteria+perclace 10.25F0.38 1.91F0.48 406F9 245F8 7.25F0.03 5.78F0.03
Cultures were incubated at 22 8C for 4 days. Results are means of triplicate independent experimentsFstandard deviation (N 1).
 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45 41

12 0.6

0.5
Remaining ClO4- (mg L-1)

10

Sugar (mg ml-1)

0.4
8
0.3
6
0.2
4
0.1
2 0
0 1 2 3 4 5 6 7
0 Days
0 0.5 1 1.5 2
Potato peel concentration (%, w/v) Fig. 3. Sugar levels in cultures during 7 days of incubation. Sterile
medium (.), sterile medium+Dechlorosoma sp. perclace (E), non-
Fig. 1. Effect of different amounts of potato peels on ClO4 sterile medium (n), and non-sterile medium+Dechlorosoma sp.
reduction by Dechlorosoma sp. perc1ace in sterile medium. Initial perc1ace (x). Results are means of triplicate independent experi-
ClO4 concentration was 10 mg L 1 and cultures were incubated for ments. Standard deviation (N 1) bars are shown.
4 days. Results are means of duplicate independent experiments.
Standard deviation (N 1) bars are shown.
3.8. pH changes in the cultures
4 days and a minor decrease was observed on day 5. A sharp decrease in culture pH from an initial
In the sterile potato peel media inoculated with value of approximately 7.2 to about 6 in one day was
Dichlorosoma sp. perclace, soluble protein in the observed in the non-sterile media. Subsequently, the
culture was stable for the first 24 h, and increased pH remained generally stable from day 1 to 7. In the
susbstantially on day 2, being relatively stable from sterile potato peel medium inoculated with perclace
day 2 to day 6. Soluble protein levels in the sterile and the non-inoculated sterile medium, the pH also
medium were generally stable with minor increases
and decreases during the incubation period.
500

12 400

10 300
Redox potential (Eh)

200
ClO4- (mg L-1)

8
100
6
0

4 -100

2 -200

-300
0
0 1 2 3 4 5 6 7 -400
Incubation time (Days) 0 1 2 3 4 5 6 7 9
Incubation time (Days)
Fig. 2. ClO4 levels in 0.5% potato peel cultures during 7 days of
incubation. Sterile medium (.), sterile medium+Dechlorosoma sp. Fig. 4. Changes redox potential of cultures during 7-day of
perclace (E), non-sterile medium (n), and non-sterile medium+ incubation. Sterile medium (.), sterile medium+Dechlorosoma
Dechlorosoma sp. perc1ace (x). Results are means of triplicate sp. perclace (E), non-sterile medium (n), and non-sterile medium+
independent experiments. Standard deviation (N 1) bars are Dechlorosoma sp. perc1ace (x). Results are means of triplicate
shown. independent experiments.
42 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45
80
70
Soluble protein (µg ml-1)
60
50
40
30
20
10
0 ClO4 reduction. Amylolytic enzymes are of great
0 1 2 3 4 5 6 7
Incubation time (Days)
Fig. 5. Soluble protein in cultures during 7 days of incubation.
Sterile medium (.), sterile medium+Dechlorosoma sp. perc1ace
(E), non-sterile medium (n), and non-sterile medium+Dechloro-
soma sp. perc1ace (x). Results are means of triplicate independent
experiments.
decreased rapidly in the first 3 days. In general,
reduction of culture pH was observed in all the potato
peel media reaching 6.0, 6.1, 5.7, and 6.4 in non-
sterile medium inoculated with and without Dichlor-
osoma sp. perclace, and the sterile medium inoculated
with and without perclace, respectively.
4. Discussion
Bioremediation is currently an attractive technol-
ogy for treatment of ClO4 contaminated water.
Various carbon substrates have been successfully
used as electron donors for ClO4 reduction. How-
ever, the cost of substrates that can be used by
microorganisms for ClO4 reduction is central to the
success and competitiveness of a bioremediation
strategy for ClO4 elimination from water. Starch is
abundant in plant materials. Mahmood et al. (1998)
demonstrated that the solid matter of the potato peel
was predominantly starch. To our knowledge, this
abundant food processing waste had not been
successfully used as a carbon source for ClO4
reduction. We have thus, explored the potential
application of starch hydrolysate and potato peel
waste for ClO4 reduction.
In our study, the starch medium inoculated with
Dichlorosoma sp. perclace plus an amylolytic bacte-
rial consortia displayed substantial reduction of ClO4
in 4 days. On the contrary, no ClO4 reduction was
observed in the starch medium inoculated with
amylolytic bacterial consortia without Dichlorosoma
sp. perclace or that inoculated with Dichlorosoma sp.
perclace without the amylolytic bacterial consortia.
Amylolytic microorganisms produce enzymes, amy-
lases, that hydrolyze starch molecules to give diverse
products including dextrins and progressively smaller
molecules (Gupta et al., 2003). The smaller molecules
are cell permeable and are available for growth and
9 importance in present day biotechnology. Although
they can be obtained from plant and animal sources,
microorganisms are the major sources of starch-
degrading enzymes with desirable properties for
industrial use (Haki and Rakshit, 2003; Gupta et al.,
2003).
Initial evaluation of white potato peel inoculated
with amylolytic bacteria only, a mixture of amylolytic
bacteria and Dichlorosoma sp. perclace, and singly
with Dichlorosoma sp. perclace revealed substantial
removal of ClO4 from water, with respective degrees
of removal of 70.2%, 90.8%, and 93.4% in 4 days.
Slightly less promising results were achieved with the
red potato peel cultures: 56%, 81.4%, and 85.3%
removal, respectively, in 4 days. Potato peels contain
low molecular carbohydrate materials and natural
amylases as well as amylolytic microorganisms that
support ClO4 reduction. Mahmood et al. (1998)
reported that potato peels contain as high as 15%
total sugar on a dry weight basis. Natural amylases
(JanNielsen et al., 1997; Witt and Sauter, 1996;
Viksonielsen et al., 1997), notably, h-amylases are
found in potatoes. Moreover, our analysis of the
natural microbial populations of potato peel waste
revealed the presence of microorganisms that can
grow on starch as the sole carbon source (data not
shown).
Analysis of the dynamics of ClO4 removal from
water using potato peels as the sole carbon source in
non-sterile and sterile media inoculated with Dichlor-
osoma sp. perclace, revealed rapid depletion of ClO4
in the non-sterile potato peel cultures with or without
the addition of the efficient ClO4 reducing bacterium,
Dichlorosoma sp. perclace. ClO4 reducing bacteria
are ubiquitous in nature. Kim and Logan (2000)
demonstrated that addition of nutrients to waste water
and soil samples was sufficient to achieve ClO4
 B.C. Okeke, W.T. Frankenberger Jr. / Science of the Total Environment 347 (2005) 35–45
reduction by indigenous microorganisms. Other
researchers have demonstrated that the ability to
reduce ClO4 is not limited to characterized mono-
cultures, but many in diverse ecosystems. Bacteria
capable of ClO4 reduction are found to inhabit a
variety of environments including rivers, sediments,
soils and waste water treatment plants, aquatic sedi-
ments, paper mill waste sludges, and farm animal
waste lagoons (Van Ginkel et al., 1995; Xu et al.,
2003; Coates et al., 1999).
Redox potential as well as availability of suitable
cell permeable carbon, are important factors that affect
ClO4 reduction. The redox potential of the non-sterile
potato peel media and inoculated sterile potato peels
media, decreased rapidly to values below 200 mV in
3 days. Generally, ClO4 reduction has been estimated
to occur below the E h of 110 mV which is the Eh
value at which the redox dye, resazurin, changes from
pink to clear (Attaway and Smith, 1993; Giblin et al.,
2000a,b; Okeke et al., 2002).
In the non-sterile potato peel media, sugar levels
remained very low. The low sugar level is
attributable to carbon assimilation for growth and
ClO4 respiration. In the sterile potato peels media,
increases in reducing sugar levels were observed
until day 4. Thereafter, it remained stable in the
non-inoculated sterile medium. Contrarily, sugar
levels in the sterile cultures inoculated with
Dechlorosoma sp. perclace, decreased substantially
between day 4 and day 7 due to growth and ClO4
respiration. The increasing concentration of reduc-
ing sugars observed in the sterilized cultures is an
indication of slow release of nutrients from the
potato peels. However, the rate of ClO4 reduction
in the sterilized cultures is less than those of the
non-sterilized cultures indicating amylolytic activity
in those cultures.
The potato peel media contained insoluble materi-
als, thus soluble protein content was used to monitor
microbial growth in the media. Protein concentration
in the two non-sterile media increased susbstantially
during the course of the incubation, but did not follow
the standard microbial growth pattern. This is possibly
due to the mixed microbial populations in the non-
sterile system. On the contrary, the soluble protein
profile of the sterilized potato peels culture inoculated
with Dechlorosoma sp. perclace displayed a standard
microbial growth curve pattern. No substantial
43
changes in protein concentration were observed in
the non-inoculated sterile cultures. General decreases
in pH to acidic levels were observed in all cultures.
Such acidification could be attributed to fermentation
of low molecular weight carbon compounds to
organic acids.
In conclusion, this paper presents promising results
on the use of purified starch and potato peel waste for
ClO4 removal from water. Carbon from potato peels
can be used to effectively sustain ClO4 reducing
bacteria. The potential use of potato peels for the
removal of oxyanions from water will not only reduce
costs of carbon substrates, but it will also aleviate the
potential environmental pollution problems from
these wastes as well as reduce disposal costs.
However, the interaction between amylolytic bacteria,
ClO4 reducing organisms, and other organisms in
mixed cultures can be complex since there is
simultaneous competition for products of starch
degradation. Thus, future studies will explore the
use of cell-free immobilized amylases to catalyze
starch hydrolysis in one reactor and sequential ClO4
reduction in a second reactor fed with carbon rich
effluents from the first reactor.
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