Biodegradation
of Recalcitrant Aliphatic, PAHs and PAS Compounds in
Liquid Culture
J.M.
Arce-Ortega and N. Rojas-Avelizapa, Biotechnology and
Bioengineering Department,
Research Center and Advanced Studies, National
Polytechnic Institute, Av Instituto Politecnico Nacional
2508, Mexico City, Mexico 07360, Tel. 30036915,
Fax. 30037705
R. Rodriguez-Vazquez, Biotechnology and Bioengineering
Department, Research Center and Advanced Studies,
National Polytechnic Institute, Av Instituto Politecnico
Nacional 2508,
Mexico City, Mexico 07360, Tel. 57473316, Fax. 57474305
During
soil remediation efforts most of the remaining and
recalcitrant compounds are branched aliphatic, PAHs and
PAS. Due to their low concentration and the lack of
standards little attention have received. Some studies
have reported the toxicity of some of them, which has
been related to their water solubility, and particle
absorption. The aim of this study was to evaluate the
capability of the native microflora of a drilling
waste-contaminated soil in liquid culture to degrade or
transform such compounds. We prepared an enrichment
culture using as inocula the native microflora of a
composted soil (120,000 mg/Kg TPHs) and the fractions
subjected to the study as sole carbon sources
(aliphatic, PAH) at a concentration of 200 mg/l.
Aliphatic and PAHs fractions were obtained from the
contaminated soil and PAS were prepared using standards
since it was not possible to get enough amount from the
soil. Each fraction was identifying by GC-MS. Three
different biodegradation kinetics were prepared in
flasks and at the appropriate time flasks were
sacrificed and evaluated for CFU/ml, CO2
production and hydrocarbon removal by GC-FID and GC-MS.
Obtained results demonstrated that branched aliphatic
hydrocarbons were removed in an extent of 90% during the
first three days of treatment which agree with the
exponential growth phase of the microbial culture.
Branched PAHs were removed at 60% during the first 18
days remaining as recalcitrant the methyl-substituted
compounds of fluorene, phenanthrene and anthracene.
Branched PAS compounds were the most recalcitrants
however, at the end of 24 days of incubation a total
removal of 90% was observed for dibenzothiophene, 60%
for methyl dibenzothiophene and no removal were observed
for the dimethyldibenzothiophene.
It is important to point out that only a few
microorganisms were able to degrade these recalcitrant
compounds.
Comparison
of Dinitrotoluene Degradation by a Mixed Culture in
Aqueous Batch System
Professor
Jan Paca, Department of Fermentation Chemistry and
Bioengineering, Institute
of Chemical Technology,
Technicka 5, CZ-166 28 Prague, Czech Republic,
Tel: +420-2-24353785
, Fax:
+420-2-24355051, Email: Jan.Paca@vscht.cz
Mr. Jiri Barta, Graduate Student, Department
of Fermentation Chemistry and Bioengineering, Institute
of Chemical Technology,
Technicka 5, CZ-166 28 Prague, Czech Republic, Tel:
+420-2-24353785, Fax: +420-2-24355051, Email:Jiri.Barta@vscht.cz
Professor Rakesh Bajpai, presenting author,
Department of Chemical Engineering, University of
Missouri, Columbia, MO 65211, USA , Tel: (573) 882 3708,
Fax: (573) 884 4940, Email:
bajpair@missouri.edu
A
mixed culture, enriched from nitrotoluenes-contaminated
aged-soil from an ammunition plant in the Czech
Republic, was used to study biodegradation of
2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene
(2,6-DNT) in aqueous media under aerobic conditions. The
study was conducted in shake flasks containing mineral
salt medium with or without additional carbon and energy
source. In first set of experiments, the medium was
supplemented either with glycerol or with succinate as
primary carbon and energy source. Two different initial
DNT concentrations (5.2 and 25 mg.L-1) were
used. When glycerol supplementation was used with 5.2
mg.L-1 initial concentrations of DNT, the
results showed total disappearance of 2,4-DNT in 4 days
and that of 2,6-DNT in 7 days. With succinate as carbon
and energy source, total disappearance of 2,4-DNT was
observed in 10 days, but the disappearance of 2,6-DNT
was poor. With
higher initial DNT concentrations, 2,4-DNT was still
removed to a higher extent than 2,6-DNT although the
reduction in the concentration of 2,4-DNT was only 50 %
after 6 days only both the carbon sources.. In addition,
metabolite(s) also accumulated in the medium.
After six months of a selection pressure by DNTs,
the mixed culture was able to degrade 2,6-DNT almost
completely, but not 2,4-DNT. Also no intermediates
accumulated in the medium.
When the dinitrotoluenes were used as the sole
source of C-, N- and energy, ~ 40 %
of each DNT
was removed within 4 days.
However, uptake of 2,4-DNT showed a lag phase and
one
unidentified intermediate accumulated in the medium.
2,6-DNT uptake did not show any lag-phase and no
intermediate accumulated in the medium.
Acknowledgement:
The work was financially supported by the Czech Grant
Agency, Project 104/01/0458
The
Effect of Aggregate and Initial Contaminant
Concentration on the Biodegradation
Rate of Total Petroleum Hydrocarbons
Ramona
Boodoosingh, Department of Civil and Environmental
Engineering, Tufts
University, Medford, MA 02155, Tel: 617-627-3211, Email:
rboodoosingh@yahoo.com
|Dr. Denise Beckles, Department of Chemistry,
University of the West Indies, St. Augustine, Trinidad,
W.I., Tel: (868) – 662- 2002 Ext 3534, Email: dbeckles@fans.uwi.tt
Ms.
Anne Marie Desmarais, Department of Civil and
Environmental Engineering, Tufts
University, Medford, MA 02155, Tel: 617-627-3211, Email:
annemarie.desmarais@tufts.edu
Dr. Christopher Swan, Department of Civil and
Environmental Engineering, Tufts
University, Medford, MA 02155, Tel: 617-627-3211, Email:
cswan@tufts.edu
Bioremediation is one of
the most cost-effective, and environmentally-friendly
methods to remediate many different types of waste
materials widely used by many private and public
entities. Though the parameters that affect the rate of
bioremediation differ for each project, some parameters
can be more easily controlled in the field than others.
For example, effective microbial action is
crucial to the success of bioremediation.
However, microbes can be detrimentally affected
by an initial contaminant concentration that is too
high. In
addition, the type of bulking agent or aggregate used
affects the ability of the mixture to retain moisture,
the amount of drainage, aeration and ultimately the
length of time for biodegradation.
A two-month field study was
conducted in Trinidad and Tobago on crude oil
contaminated waste.
This paper will look at the effect of two
parameters, initial concentration and type of bulking
agent, on the rate of biodegradation of Total Petroleum
Hydrocarbons (TPH) in the oily waste being treated in
this tropical environment.
A nutrient-supplying bioremediation agent was
used to enhance the bioremediation process.
The results of this study do suggest that the
development, operation, and maintenance of
bioremediation projects require a significant amount of
planning. The results also suggest that the use of a bulking agent can
assist in effective bioremediation of heavy crude oil
wastes in tropical environments.
Effect
of Bacteria Augmentation on Aromatic and Asphaltenic
Fraction Removal in Solid Culture
E.
Cervantes-Gonzalez, L.I. Rojas-Avelizapa, R.
Cruz-Camarillo, National School of Biological Sciences,
Carpio y Plan de Ayala S/N Col. Casco de Santo
Tomas, ZP 11340, Mexico
City, Mexico,
Tel: (52)53412343, Email: elcege@hotmail.comN.G.
Rojas-Avelizapa, Instituto Mexicano del Petróleo,
Eje Central Lazaro Cardenas 152, ZP
07730. Mexico City, Mexico, Tel: (52) 30036915, Fax:
(52) 33037705
Soil
contaminated by petroleum is a pervasive problem in
Mexico mainly in the Southeast (Tabasco and Veracruz)
which has provoked a serious damage of the ecosystem.
Biological treatment of hydrocarbon-contaminated soil is
considered to be a relatively low-cost and safe
technology, however, exhaustive treatability studies
must be done to find those nutritional or environmental
conditions that improve hydrocarbon biodegradation.
Another important limitation to apply biological
treatments in Mexican soils is the high level and age of
the contamination. This study examines the biological
treatment technology to clean up petroleum-contaminated
soil with special emphasis in bioaugmentation and
nutrimental conditions. The soil used for this study was
an acidic soil (pH 3) containing 220,000 mg/Kg TPHs and
a native population of 2.0x105 CFU/g.
Previous work demonstrated that only two bacteria
were involved in the aromatic and asphaltenic fractions
in liquid culture thus its bioaugmentation was
investigated. Three experimental sets were prepared a)
C/N/P ratio of 100/11/1 and 3% of inocula b) C/N/P ratio
of 100/11/1 without inocula and c) controls HgCl2
sterilized. Microcosms were prepared in 125 mL serum
bottles containing 25 g of soil; moisture was adjusted
to 36% and incubated at 28°C during 74 days. During all
incubation period aromatic and asphaltenic fraction
removal, CO2 production, C/N and C/P ratios,
moisture, total bacteria and pH were monitored each 7
days. The results showed that CO2 production
was higher in those microcosms bioaugmented. Removal of
aromatic and asphaltenic fractions was enhanced at an
extent of 11% (24,200 mg TPHs/Kg) respect to those
microcosms not bioaugmented during the first 38 days of
treatment. Bacteria had its growth exponential phase
during the first 30 days, which correlate with both
fraction removal. No important changes were observed for
C/N ratio, moisture and pH, however C/P ratio increases
during the first 7 days of treatment.
Biodegradation
of Diuron and Staple by White-Rot and Soil Fungi
R.
M. A. Gondim-Tomaz, T. T. Franco, and L.R. Durrant, Food
Science Department - Food Engineering Faculty, Campinas
State University, Campinas –SP, Brazil
The
aim of this work was to isolate fungi from soil treated
with the two pesticides, Diuron and Staple 280CS, and to
screen for those strains able to degrade the highest
levels of these two compounds. Twenty-four ligninolytic
white-rot fungi were also used in the preliminary
screen. A solid medium containing 1X, 10X or 100X of the
pesticides dosages indicated for field application was
used as sole carbon sources. Thirteen strains were
selected: Pleurotus
sp BCCB 507, P. sp CCB 068, P. sp. 016, Agaricus
campestris, Phanerochaete
chrysosporium ATCC 24725 and the soil isolates
DP24e, DP24o, DRP02n, SP16a, SRP 17c, SRP17g and SRP20e.
These fungi were grown in liquid medium containing
minerals and yeast extract for three days when 25 ug/ml
of Diuron or 10 ug/ml of Staple were added to the
culture flasks and cultivation was carried out for up
to14 days. Triplicate samples were collected and the
supernatants were used for the determination of
ligninolytic activities
(spectrophotometric assays) and
degradation (by HPLC).
When
Diuron was used the strains Pleurotus
sp BCCB 507 (60.70% - 7th day), P.
sp CCB 068 (80.75% - 10th day), P. sp. 016 (58.60% - 7th day), and the soil isolates
SRP17g (65.46% - 10th day), SRP 17c
(67.60% – 14th day) and SRP 20e (62.20% -
10th day). MnP was the predominant enzyme
produced by all strains.
When
Staple 280 CS was used for growth only three strains
were able to degrade it: Pleurotus
sp BCCB 507 (14.25% - 7th day), A.
campestris (32.90%-14th day) and P.
sp. CCB 068 (53.20% - 7th day). MnP was
also the predominant ligninolytic enzyme produced. The
more complex chemical structure of the pesticide Staple
may be responsible for the lower levels of degradation
and of strains able to attack it.
Considering
both pesticides Pleurotus
sp CCB 068 was the best strain among all the strains
tested. It is important to mention that the highest
levels of enzymes activities were produced by this
strain, regardless of the pesticide.
Biodegradation
of PAHs in Soil by
Two Deuteromycete Fungi
A.R.
Clemente and L.R. Durrant, Food Science Department -
Food Engineering Faculty, Campinas State University,
Campinas –SP, Brazil
The
fungal strains used is this work, namely, 984 and 1040,
were a gift from Fundação Tropical André Toselo, and
were isolated from soil samples collected at the
Jureia-Itatins Ecological Reserve. Following
microscopically examinations these strains were
classified as Aspergillus sp. (984) and Verticillium
sp. (1040). The degradation of PAHs in soil contaminated
with 5 mg
naphthalene/g soil; 1,0 mg anthracene/g soil or 0,5 mg
pyrene and/or benzo[a]pyrene/g soil, was verified. These
strains were grown in
wheat bran:water for 3 days and inoculated into
two portions of soil, sterilized and non- sterilized,
and cultivated for 2, 4, 6, and 8 weeks, when the PAHs
were extracted from the soil and the degradation was
determined by HPLC. Degradation varied with the strains
and also with the soil treatment. The best degradation,
in sterilized soil was obtained after 8 weeks of growth
for the two strains: naphthalene (64.50-65.43%),
anthracene (77.35-85.83%) and pyrene (73.01-78.78%).
When benzo[a]pyrene was used the best degradation shown
by strain 984 was 89.62% in six weeks and by the strain 1040
78.06% in eight weeks. In non-sterilized soil the
strains exhibited lower growth and degradation than in,
with the exception
for benzo[a]pyrene (82.3-82.6%). Our results indicate
that these two fungal strains
have potential for application in the
bioremediation of soils contaminated with PAHs.
We
acknowledge FAPESP for financial support.
In-situ
Bioremediation of Pesticide Impacted Soil at the THAN
Superfund Site, Montgomery, Alabama
David Raymond, M.Sc., Adventus Remediation Technologies,
1345 Fewster Dr., Mississauga, Ontario, Canada L4W 2A5
Tel: 905-273-5374 x. 224, Email: david.raymond@adventustech.com
Steven Gable,
M.Sc, P.Eng., P.E., MBA, rmc, Adventus Remediation
Technologies, 1345 Fewster Dr., Mississauga, Ontario,
Canada L4W 2A5, Tel: 905-273-5374 x. 228, Email: steven.gable@adventustech.com
Geoffrey Bell, M.Sc., Adventus Remediation Technologies,
1345 Fewster Dr., Mississauga, Ontario, Canada L4W 2A5,
Tel: 905-273-5374 x. 225, Email: geoff.bell@adventustech.com
Alan Seech, Ph.D., Adventus Remediation Technologies,
1345 Fewster Dr., Mississauga, Ontario, Canada L4W 2A5,
Tel: 905-273-5374 x. 221, Email: alan.seech@adventustech.com
Todd Slater, B.Sc., ATOFINA Chemicals, Inc., 2000 Market
St., Philadelphia, PA 19103-3222, Tel: 215-419-5824,
Email: todd.slater@atofina.com
Further
to the results of a pilot-scale demonstration completed
in May 2001, the USEPA selected Adventus Remediation
Technologies’ patented DARAMEND®
bioremediation for full-scale treatment of
pesticide-impacted soils at the T.H. Agriculture and
Nutrition (THAN) Superfund Site (Site) in Montgomery,
Alabama. To
our knowledge, this represents the first ever,
full-scale, in-situ, solid-phase application of
bioremediation to pesticide-impacted soils.
In particular, Site soils were impacted with
Toxaphene, DDT, DDD, and DDE.
Treatment results correspond closely to those
seen during the pilot-scale demonstration, during which
DDT and Toxaphene concentrations (the only compounds
that exceeded the Performance Standard) were reduced
from 317 mg/kg and 168 mg/kg to 24 mg/kg and 26 mg/kg
respectively, during 168 days of active treatment.
To date, after approximately 75 days of active
full-scale treatment, Toxaphene, DDT, DDD, and DDE
concentrations have been reduced by 63%, 70%, 27%, and
32%, respectively. Relatively high initial DDT concentrations resulted in the
transient accumulation of DDD in some sampling zones. Reductions in DDD concentrations in these sampling zones are
now expected to accelerate, due to the sharp reduction
in DDT concentrations.
This pattern is consistent with that observed
during bench- and pilot-scale studies conducted on this
and other pesticide-impacted sites.
It is anticipated that all Performance Standards
for the Site will be reached following 50 to 75 days of
further treatment.
Results from the completed project will be
presented.
DARAMEND
is a registered trademark of Adventus Intellectual
Property Inc.
Enhanced
Aerobic Bioremediation of Petroleum Hydrocarbons using
Permeox® Plus
Scott
M. Hulseapple, M.S.,
URS Corporation, 646 Plank Road, Suite 202, Clifton
Park, New York 12065, Tel: 518-688-0015, Email:
scott_hulseapple@urscorp.com
Stephen B. LeFevre, PG, CPG, M.S., URS Corporation, 646
Plank Road, Suite 202, Clifton Park, New York 12065,
Tel: 518-688-0015, Email: stephen_lefevre@urscorp.com
Chuck Elmendorf , B.S., Panther Technologies, Inc., 220
Route 70 East, Suite B, Medford, New Jersey, 08055, Tel:
609-714-2420, Email: celmendorf@panthertech.com
Peter J. Palko, P.E., CHMM , B.S., Panther Technologies,
Inc., 220 Route 70 East, Suite B, Medford, New Jersey,
08055, Tel: 609-714-2420, Email: ppalko@panthertech.com
This
presentation will detail the results of a full-scale in-situ enhanced bioremediation program targeting petroleum
contamination at a school bus maintenance garage in
Otego, New York. Leaky USTs and impacted soils were
removed in 1998. However,
groundwater impacts in the sandy aquifer remained.
In-situ
enhanced bioremediation was proposed as a cost-effective
alternative to conventional ex-situ
remedial methods. Furthermore,
the current site use limited the available space for
above ground infrastructure associated with conventional
ex-situ
remedial methods.
It
is well documented that the release of oxygen in the
subsurface environment is known to enhance the
biodegradation of petroleum hydrocarbons.
PermeOx® Plus is a form of calcium
peroxide designed to release oxygen to enhance the
biodegradation of petroleum hydrocarbons in soil.
PermeOx® Plus provides oxygen through
a reaction of the specialty formulation of calcium
peroxide and water:
CaO2
+ 2H2O→Ca(OH)2 + H2O2
2H2O2→O2
+ 2H2O.
The
treatment area was approximately 200 feet by 80 feet
with an 8-foot thick contaminated zone.
Total BTEX mass in July 2002 was 4.7 kg.
In July, 2002 PermeOx® Plus was
injected using a direct-push drill rig at 107 locations
within the plume area.
In addition, Waste Stream Technology’s Bioblend™
M-4, a blend of petroleum degrading bacteria and
nutrients, was injected into the contaminant plume.
Within 5 months, total BTEX mass within the plume was
reduced to 1.7 kg in December 2002.
A second PermeOx® Plus injection was
conducted in a 140 feet by 70 feet area at 55 locations.
Subsequent monitoring events are scheduled to
monitor the progress of the remedial program.
The
presentation will discuss the use of PermeOx®
Plus to introduce oxygen into the contaminated
groundwater plume. The presentation will also discuss the overall effectiveness
of the remedial program for reducing dissolved-phase and
sorbed-phase petroleum hydrocarbon concentrations to
levels that will achieve site closure.
Degradation
of 4-Aminobenzene Sulphonate by a Newly Isolated
Bacterial Culture
Poonam
Singh, Biotechnology Laboratory, Department of
Chemistry, I.I.T, Kanpur- 208016, India
, Tel:
+ 91-512-2597104, Fax: + 91-512-2597436, Email: singhpoonam3012@rediffmail.com
Sami Sarfaraz, Biotechnology Laboratory, Department of
Chemistry, I.I.T., Kanpur- 208016, India
, Tel:
+ 91-512-2597104, Fax: + 91-512-2597436, Email: samisarfaraz@hotmail.com
L.C. Mishra, Department of Life Sciences, C.S.J.M.
University, Kanpur 208016, India
Leela Iyengar, Biotechnology Laboratory, Department of Chemistry, I.I.T., Kanpur- 208016,
India, Tel:
+ 91-512-2597104, Fax: + 91-512-2597436, Email:
leela@iitk.ac.in
4-Aminobenzene
sulfonate (4-ABS) is the building block for the
production of many azo dyes, pharmaceuticals, pesticides
and other consumer products. Consequently it is
synthesized and released into the environment in large
quantities and thus a major pollutant of surface waters.
Aryl sulphonates are generally recalcitrant to microbial
degradation. Only few reports are available on the
utilization of 4-ABS, either as a sole or mixed carbon
and energy source, by defined or pure bacterial
cultures. In this communication, we report the studies
on 4-ABS degrading bacterial isolate strain PNS2. Strain
PNS2 was isolated from the enrichment culture developed
from the inoculum derived from large scale activated
sludge treatment unit receiving Kanpur City sewage.
Cells of the strain PNS2 were gram negative rods and
catalase +ve. Base sequence of 16 rDNA revealed an
affiliation with Agrobactrerium
tumefaciens.
Strain
PNS2 utilized 4-ABS as the sole carbon and energy source
on mineral salt medium, without any specific addition of
vitamins. It could also serve as the source of nitrogen
and sulfur for the growth of the organism. Exponential
growth was observed upto a concentration of 1000 mg/l
and shortest doubling time during the growth on 4 ABS
was around 7 hr. Strain PNS2 was highly specific for the
position of amino group substitution as neither 2- or
3-aminobenzene sulphonates could serve as growth
substrates. Degradation of 4-ABS was accompanied by the
release of ammonia, (around 80% ABS degraded) as well as
sulphate. No aromatic metabolites of 4-ABS could be
detected in the culture filtrate either during growth
phase or with dense cell suspensions. Total organic
carbon analysis of the culture filtrate showed that
4-ABS was completely mineralised. Preliminary studies
have shown the presence of a plasmid in the strain PNS2.
Studies
on organisms degrading aminobenzene sulphonates,
metabolic pathways specific enzymes and the genes
involved in the degradation can lead to their
application for the removal of these environmental
pollutants.
Rapid
Biological Treatment of Residual DNAPL with Slow Release
Electron Donor HRC-X™
Stephen
S. Koenigsberg,
Ph.D., Regenesis, 1011
Calle Sombra, San Clemente, CA
92672,
Tel: 949-366-8000,
Fax: 949-366-8090,
Email: steve@regenesis.com
Anna Willett, 1011 Calle Sombra, San Clemente, CA
92672, Tel:
949-366-8000, Fax: 949-366-8090, Email:
anna@regenesis.com
The
use of in situ
bioremediation to stimulate the rapid dissolution,
desorption, and biodegradation of residual DNAPL has
been demonstrated in the laboratory and in
well-documented field studies.
Biodegradation of dissolved-phase contaminants
increases the partitioning and subsequent biodegradation
of residual DNAPL to the aqueous phase by (1) increasing
the concentration gradient and driving force for
dissolution and desorption and (2) increasing the
overall solubility of the DNAPL by production of
hydrophilic daughter products.
Specifically,
the application of the slow release electron donor
substrate, Hydrogen Release Compound-Extended Release (HRC-X™),
has been successful in remediating high concentrations
(>100 mg/L) of chlorinated ethenes, like PCE and TCE
in residual DNAPL environments.
In situ
bioremediation with HRC-X is a low-cost method for
residual DNAPL removal and avoids the costly and lengthy
assessment associated with defining the exact location
of the dispersed residual DNAPL.
HRC-X
is a highly concentrated electron donor for
bioremediation and has a field longevity of
approximately 3 years.
Injection of HRC-X directly into the general
residual DNAPL area of a contaminated aquifer results in
the continuous release of lactic acid and fermentation
of the lactic acid to hydrogen in, surrounding, and
downgradient of the injection area.
Hydrogen from HRC-X is used as an electron donor
for reductive dechlorination of chlorinated ethenes,
which results in dissolution of residual DNAPL and
desorption of sorbed contaminants.
This
presentation includes a description of HRC-X, as well as
the mechanisms by which chlorinated ethene contaminants
are dissolved, desorbed, and degraded.
Case histories describing the successful field
application of HRC-X for bioremediation of residual
DNAPL will be presented.
Enhanced
Reductive Dechlorination – A Broader Perspective
Suthan
S. Suthersan, ARCADIS G&M, 3000 Cabot Blvd.W., Suite
3004, Langhorne, PA 19047, Tel: 215-752-6840, Fax:
215-752-6879
Frederick C. Payne, ARCADIS G&M, 25200 Telegraph
Road, Southfield, MI 48034, Tel:
248-936-8000 Fax: 248-936-8111
Denice K. Nelson, ARCADIS G&M, 420 North Fifth
Street, Suite 1035, Minneapolis, MN 55401, Tel:
612-339-9434, Fax: 612-336-4538
Enhanced
reductive dechlorination of chlorinated compounds has
been applied via in situ engineered anaerobic systems in
increasing numbers over the past decade.
Knowledge gained from these field applications, combined
with academic literature, allows us to have a broader understanding
of the technology. Microbial communities can be
engineered through manipulation of aquifer inputs
creating in-situ reactive zones. Over a short
segment of the groundwater flow path, the microbial
biomass is shifted to a succession of facultative
degraders and fermenters, sulfate reducers, acetogens
and methanogens (termed the anaerobic metabolic
cascade). At low to moderate electron donor loading
rates, the aquifer microbial continuum is dominated by
species that produce C-1 compounds and acetate as the
fermentation products. When electron donor loading increases to high levels a
significant portion of the electron donor flows into
pathways that generate fatty acids. Bacteria from
several points in the metabolic cascade have been shown
to reductively dechlorinate halogenated compounds.
Reductive dechlorination occurs by two basic
mechanisms: metabolic – bacteria utilize chlorinated
compounds as electron acceptors, and cometabolic –
enzymes and cofactors produced by bacteria can
dechlorinate solvents.
Analysis of chemical thermodynamics shows energy
gained from chlorinated ethenes as electron acceptors is
sufficient to support growth. Although some dechlorinating species do not degrade vinyl
chloride at meaningful rates, this is not due to
thermodynamic limitation.
Biostimulation using late-cascade compounds such
as lactate and methanol by-pass much of the productive
dechlorinating communities. The repeated ability to
dechlorinate with high carbon loading rates and without
bioaugmentation suggests the importance of these groups.
These observations were developed from
the authors’ research and published reports of field
applications and lab research.
Data from five case studies is presented to
illustrate the general principles of reductive
dechlorination.
Aerobic
Mononitrotoluene Biodegradations in Batch and Continuous
Reactor Systems
Jan
Paca, Department of Fermentation Chemistry and
Bioengineering, Institute of Chemical Technology,
Technicka 5, CZ-166 28 Prague, Czech Republic,
Tel: +420-2-24353785, Fax: +420-2-24355051,
Email: Jan.Paca@vscht.cz
Jiri Barta, Department of Fermentation Chemistry and
Bioengineering, Institute of Chemical Technology,
Technicka 5, CZ-166 28 Prague, Czech Republic, Tel: +420-2-24353785, Fax: +420-2-2435505, Email:
Jiri.Barta@vscht.cz
Rakesh Bajpai, Department of Chemical Engineering,
University of Missouri, Columbia, MO 65211, Tel:
573-882 3708, Fax: 573-884 4940, Email: bajpair@missouri.edu
A
mixed culture enriched from aged nitrotoluenes-contaminated
soil from an ammunition plant in the Czech Republic was
used to study biodegradation of 2-, 3-, and
4-nitrotoluene in aqueous solution under aerobic
conditions. The degradation in batch system was carried
out with free cells (mixed culture) in shake flasks
containing basal salt medium. The mononitrotoluenes (MNT)
served as a sole carbon, energy, and nitrogen source for
the cell growth. Here, a degradation of all the MNT’s
and their mixture was tested. The NT removal rate and
efficiency was evaluated. Also the kinetics of 4-NT
degradation was determined. In the second part, similar
experiments were performed in a continuously operated
packed bed reactor with the cocurrent
water-air upflow mode. The cells were immobilized
on expanded slate used as the packing material. The
start-up period and degradation characteristics for 4-NT
are described. The degradation rates and efficiencies
are evaluated and compared. During this long-term
experiment lasting 8 months also changes in the biofilm
composition, viability count of immobilized and free
cells, and microscopy observations (SEM) of a biofilm
structure were estimated and the results are discussed.
A
complete removal of all the MNT’s in all the batch and
continuous experiments has been achieved (HPLC
determination).
Acknowledgement:
The
work was financially supported by the Czech Grant
Agency, Project 104/01/0458
Effectively
of Hydrocarbons Contaminated Soils Biodegradability Test
Evaluated in Different Mexican Soils
Martha
E. Ramirez,
Berenice Zapien and Luis C. Fernandez, Instituto
Mexicano del Petroleo, Eje
Central Lazaro Cardenas # 152, Mexico City, Mexico,
A.P.14-805, C.P. 07730, Tel: (5255) 3003-6915 Fax:
(5255) 3003-7705, Email: mrislas@www.imp.mx
As
a preliminary proof of biological hydrocarbonoclastic
activity and the feasibility to apply a biological
technique for the treatment of oil contaminated soils,
the evaluation of biodegradative capacity of
contaminated soils at laboratory scale is necessary.
Generally, the biodegradability tests, in natural
conditions of the contaminated soils, are very slow. It
has been demonstrated that biostimulation, control of
water content and aeration increase the rate of
hydrocarbon biodegradation, lead to a faster response.
The aim of this work was to prove a biodegradability
test for oil contaminated soils, which was established
previously in our laboratory, for four Mexican soils
contaminated with petroleum hydrocarbons or drilling
wastes. The biodegadability tests were performed in
microcosms containing 20 g of soil (dry weight)
adjusting C:N:P ratio to 100:1.7:1.2 with NH4NO3
and KH2PO4 , water content to 3.5 times the holder capacity, temperature 30 °C
and agitation 100 rpm. Two controls were performed:
non-stimulated soil and sterile stimulated soil (sodium
azide, 2%). The parameters measured were pH, total
petroleum hydrocarbons (TPH), gas chromatography, total
carbon, total nitrogen, phosphorous, heterotrophic and
hydrocarbon-degrading microorganisms, microbial
diversity and variation in time by DGGE and CO2 evolution.
The four soils tested shown a response of degradability
6 to 9 time faster than non-stimulated systems. Also,
these soils had higher CO2 production than
control soils. However, the microbial counts were
constant during incubation in both stimulated and
non-stimulated soils. The microbial population is been
analyzed by DGG technique. This study shows that the
four soils tested had hydrocarbon biodegradation
activity and the biodegradability test is feasible to be
applied in different contaminated soils, reducing the
time to obtain a desired response.
Subsurface
Microbial Adaptation to Chemical Stressors
Rachelle R. Rhodes, Charles E. Via Department of Civil and
Environmental Engineering, Virginia Polytechnic
Institute and State University, 418 Durham Hall,
Blacksburg, VA 24061, Tel: 540-231-5293, Fax:
540-231-7916, Email: rheck@vt.edu
Denise Gillam, Department of Civil and Environmental
Engineering, University of Cincinnati, 765 Baldwin Hall,
2624 Clifton Avenue, Cincinnati, OH 45221-0071, Tel:
(513)556-2498, Email: dgillam@vt.edu
Irina Chakraborty, Charles E. Via Department of Civil
and Environmental Engineering, Virginia Polytechnic Institute and State University, 418
Durham Hall, Blacksburg, VA 24061, Tel: 540-231-5293,
Fax: 540-231-7916, Email: ichakrab@vt.edu
Ann Stevens, PhD., Department of Biology, Virginia
Polytechnic Institute and State University, 4036 Derring
Hall, Blacksburg, VA 24061, Tel: 540-231-9378, Email:
ams@vt.edu
Paul Bishop, PhD., Department of Civil and Environmental
Engineering, University of Cincinnati, P.O. Box 210071,
Cincinnati, OH 45221-0071, Tel: 513-556-3675, Email:
paul.bishop@uc.edu
Nancy G. Love, PhD., Charles E. Via Department of Civil
and Environmental Engineering, Virginia Polytechnic
Institute and State University, 418 Durham Hall,
Blacksburg, VA 24061, Tel: 540-231-3980, Fax:
540-231-7916,Email: nlove@vt.edu
Natural attenuation in contaminated subsurface
environments by biofilms can be an important factor in
contaminant removal and may be influenced by how the
biofilm community reacts to the contaminating chemicals.
Subsurface
microbial communities may activate adaptive strategies
to xenobiotic influxes via catabolic and/or molecular
stress response mechanisms.
It is hypothesized that both the concentration
and nature of xenobiotic stressors influences microbial
adaptation to plumes in contaminated subsurface
environments. Laboratory flow-through sand columns containing sacrificial
flow cells have been constructed and are currently being
operated to determine if a known stress response, called
the glutathione-gated potassium efflux (GGKE) mechanism,
manifests into a detectable community adaptive response.
GGKE is a mechanism that protects cells against
damage by thiol-reactive electrophilic compounds.
In wastewater, the GGKE response to electrophilic
chemical perturbations has been found to cause
deflocculation. Similarly,
the GGKE system is expected to cause biofilm detachment
when non-catabolized electrophilic compounds are
present. When
compounds are present that can be catabolized and
activate GGKE, it is expected that biofilm disruption
will be minimized.
Three contaminants will be tested during this study: benzene,
which is degradable and non-electrophilic; cadmium,
which is non-degradable and electrophilic (thiol-reactive);
and pentachlorophenol, which is degradable and
electrophilic. Contaminant
concentrations starting at 2 mg/L will be ramped to 10
mg/L over a seven month time period.
At regular intervals, pH, dissolved oxygen, total
organic carbon (TOC), potassium, and contaminant
concentrations will be measured along the length of the
sand column. Flow
cells connected along the length of the column,
containing column inoculum, will also be sacrificed in
regular intervals to determine levels of volatile
solids, protein, carbohydrate, lipid phosphate,
glutathione, and bacterial community profiles.
Bacterial community change over time will be
determined using temporal temperature gradient
gel electrophoresis.
