Relative Quantitative of Gene Expression during Biodegradation to Select Reliable Reference Genes
Qing Chang^1,2, Dr, ¹Graduate School of Environmental and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan , Tel: 81-45-339-4369, Fax: 81-45-339-4354, Email: JKL1255@hotmail.com
²College of Environment and Biology Engineering, Chongqing Technology and Business University, Xuefu Ave, Nan’an District, Chongqing 400067, China
Takashi Amemiya, PhD. Professor, Graduate School of Environmental and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan , Tel: 81-45-339-4353, Fax: 81-45-339-4353, Email: amemiyat@ynu.ac.jp
Kiminori Itoh, PhD. Professor, Graduate School of Engineering, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan , Tel: 81-45-339-4354, Fax: 81-45-339-4354, Email: itohkimi@ynu.ac.jp

Many microorganisms can use toxic hydrocarbon as the sole source of carbon and energy under aerobic condition to grow. Real-time PCR is a common molecular biological method for quantitative gene expression in environment. In determining relative gene expression by quantitative measurements of mRNA levels, reference genes are essential because there are differences in cellular input, RNA quality, and RT (reverse transcription) efficiency between samples. Thus, using valid reference genes is a prerequisite for accurate gene relative quantification. The ideal reference genes should be expressed relatively constant level between samples and different experimental conditions. However, different environmental conditions might also affect the expression of reference genes.

In this study, Pseudomonas putida mt-2 biodegraded p-xylene in M9 mineral medium to analyze a panel of five candidate reference genes (rpoN, rpoD, dbhA, phaF, 16S rRNA) and to determine the expression stabilities of five genes and to calculate a reliable normalization factor using geNorm software. Using the target gene, xylA, as a model degrader, illustrated the need of proper reference genes for normalization. 

According to their expression stability, geNorm software analysis revealed that the rpoN, rpoD, and 16S rRNA genes were suitable reference genes, while the phaF and dbhA genes showed unstable expression genes. Using an unstable gene as a normalizer may not only show over- or underestimation target gene expression, but also the delay in maximal gene expression, and the increasing gene expression without inducer. The work suggests that the reliability of reference genes in gene profiling studies of dynamic environment is important.

Biodegradation of Aromatic Compounds and PAHs by Halophilic Prokaryotes
Lucia R. Durrant, Maricy Bonfá, Francine Piubelli, Departamento de Ciência de Alimentos, FEA, Universidade Estadual de Campinas, Campinas, SP, Brazil
Sara Cuadros-Orellana, Instituto de Tecnologia e Pesquisa, Universidade Tiradentes, Aracaju – SE, Brazil  

It is widely known the ability of microorganisms to metabolize xenobiotics in the environment and their study has received much attention due to the environmental persistence and toxicity of these compounds. After several works published in the field, a wide variety of microorganisms in pure and mixed culture under both aerobic and anaerobic conditions capable of degrading pollutants it is now known. Among prokaryotes, haloarchaea and halobacteria are known as a group with a potential for bioremediation. Geological formations, such as petrol reserves, are associated with salty environments. Furthermore, for every barrel of oil produced, approximately 10 barrels of brackish or saline water is generated. Presently over 5 billion gallons a day of produced water is generated in the US. The saline content is high and the organic composition is very complex, varying widely in concentration. Thus, the treatment and disposal of produced waters is a challenging task, since the impact and toxicity on soils, vegetation, surface water and shallow ground water is high. That is the reason why halophilic prokaryotes can be considered as a good environmental tool for bioremediation in extremely salty environments or polluted deserts. The work developed in our laboratory in the last few years has been related to the isolation of halophilic prokaryotes able to metabolize aromatic compounds. For that various hypersaline location were sampled. Twelve strains able to grow on 1,2 benzoanthracene, 44   strains able to use p-hydroxybenzoic acid and one strain able to use phenol, as the sole carbon sources were isolated. After some studies carried out with some of these isolates we have  detected activity on the degradation of some PAHs such as of naphthalene, anthracene, fenanthrene, pyrene or 1,2-benzoanthracene, when cultivated with or without yeast extract (50 to 70% degradation). Some strains have also been grown on aromatic acids, giving some positive results on the degradation of p-hydroxybenoic acid. These strains were also used on biodegradation studies of hydrocarbons in produced waters showing promising results (COD reduction up to 73%).

Degradation of Azo Dyes by Bacteria Isolated from a Textile Industry Effluent
Elisangela F. Dias, Fábio G. Dias, Isis S. Silva and Lucia R. Durrant, State University of Campinas, Sao Paulo, Brazil, Email: durrant@fea.unicamp.br

Azo dyes are synthetic organic compounds widely used in industrial processes. The decolourization of azo dye by microorganisms starts by a reductive cleavage of azo bonds under anaerobic conditions, generating toxic amines. The main objective of this study was to verify microbial biodegradation of azo dyes RBN 198 and RB 220 by strains isolated from effluent of textile industries (Itatiba-SP, Brazil).Under in microaerobic conditions followed by agitation. The degradation of dyes was evaluated through spectrophotometer and COD reduction analyses. The formation of by-products using HPLC were also determined. Bacteria identifications were performed by sequencing of rRNAs 16S gene. Staphylococcus arlettae degraded completely the RBN 198 and RB 220 after 24 hs and 168 hs and COD reached 41 and 68%, respectively. Micrococcus luteus also degraded completely RBN 198 and RB 220 dye, while COD reduction was 78% for both dyes. These bacteria showed capacity to metabolize degradation products from the breakdown of dyes under agitation and have potential application in bioremediation. Currently, we are focusing on the formation of amines by reduction of azo dyes, as well as its  degradation under aerobic conditions.

Bioremediation of Soils Contaminated with Transformer Oil in Brazil
Satya Ganti, Sarva Bio Remed, LLC, 11 North Willow Street, Trenton, NJ, 08608, Tel: 609-695-4922, Fax: 419-710-5831, Email : satyaganti@sarvabioremed.com
Eduardo Trindade, LACTEC, P. O. Box 19067, Curitiba - Parana – Brazil, CEP 81531.980, Tel: 011-5541-3361-6270, Email : trindade@lactec.org.br
Paulo Fernandes, SDM do Brazil Ltda, Rua Rocha, 187 cj 124, Sao Paulo, SP – Brazil, CEP 01330-000, Tel: 55-11 3266-7886, Email : paulo@sdmdobrasil.com.br

COPEL is the state owned utility company supplying electricity to the entire state of Parana - Brazil.  One of the main tasks of the company is to maintain and replace electricity transformers throughout the State.  Transformers are received by the central facility for replacing and recharging. The company has a large stock of mineral transformer oil for these purposes since 1964 and the factory soil is heavily contaminated. The site represents about 18,400 square meter area that requires to be cleaned up from contamination. This area adjoins an old stream bed of River Atuba which is currently the limit between the property of the Utility Company and the urban area. The edges of River Atuba are classified as "Area of Permanent Protection" (APP), and hence required stringent environment conditions for cleanup. It was decided to use AgroRemed for achieving the cleanup without contaminating uncontaminated areas and river waters.  The protocol for treatment was to pump and treat the contaminated water before discharge.  The TPH values at the hot spots were reduced from original 700,000 ppm to less than 400 ppm. Data till end of 2007 are presented in the paper.

Surfactant Enhanced Bioremediation of F3 and F4Contaminated Soils
George A. Ivey, B.Sc., CEC, CES, CESA, Ivey International Inc., PO Box 706, Campbell River, BC  V9W 6C9, Canada.  Tel:  250-923-6326, Fax:  250-923-0718, Email:  budivey@island.net
Dan Stangroom, Remediation Division Manager, Veolia Environmental Services, 64 Allingham Street, Condell Park, NSW, 2200, Australia, Tel:  +61(0)2 8709 9509, Fax:  +61(0)8709 9534, Email:  dan.stangroom@veoliaes.com.au

This paper will focus on the application of non-ionic surfactants to improve the “bio-availability” of higher molecular weight (HMW) compounds such as F3 (C16-C34) and F4 (C34-C50) heavy-end petroleum hydrocarbons and polycyclic aromatic hydrocarbons (PAH), among others, for microbial bioremediation.

During the past decade, much discussion has centered on the unavailability of absorbed compounds to soil microorganisms. It is generally now assumed that desorption and diffusion of bound contaminants to the aqueous phase is required for microbial degradation (W.P. Inskeep, J.M. Wraith, C.G. Johnston, Hazardous Substance Research Center, 2005).

It had been well established in literature that >90% of LNAPL and DNAPL contaminants prefer to sorbed (i.e., absorbed or adsorbed) on surfaces such as soil and bedrock, versus being in the dissolved water-phase.  The sorption of contaminates to substrates is often considered the principal limiting factor affecting many remediation technologies (i.e., pump and treatment, oxidation, bioremediation, etc.). This fact limits the effectiveness of many bioremediation processes, as the targets contaminants are not “bio-available”.  Surfactants enhance bioremediation involves the use of surfactants to desorb the contamination and significantly improve the bio-availability of many recalcitrant compounds. In doing so this allows for their improved microbial mineralization during the in-situ and ex-situ applications.

Investigation of Amendments on the Biodegradation of Perchlorate
Student Presenter
Agnes B. Morrow, Jackson State University, Environmental Science MS Program/ U.S. Army Corps of Engineer-Environmental Research and Development Center, 3909 Halls Ferry Road, Vicksburg, MS 39180, USA, Tel: 601-634-2392, Fax: 601-634-2742, Email: agnes.b.morrow@us.army.mil
Victor Medina, Ph.D., U.S. Army Corps of Engineer-Environmental Research and Development Center, 3909 Halls Ferry Road, Vicksburg, MS 39180, USA, Tel: 601-634-4283, Fax: 601-634-3518, Email: victor.f.medina@us.army.mil  

The perchlorate anion (ClO4^-) is extremely soluble in both water and polar organic solvents. Consequently, it is highly mobile in groundwater under typical environmental conditions. Perchlorate is very slow to react and very persistent in the environment.  Perchlorate affects thyroid activity in many people and may have other, unknown health effects.  Therefore, the EPA placed it on the Contaminant Candidate List (CCL) in 1998. 

The primary route of exposure of perchlorate to the general public is through drinking water contaminated with perchlorate. Another possible form of exposure would be by eating vegetables that have been irrigated with water that contains perchlorate. People who live near areas where perchlorate has been used, tested, produced, or disposed of may be exposed to perchlorate in their drinking water or food. Perchlorate has been found at a number of Department of Defense (DOD) sites.  The DOD requires cost effective methods to treat perchlorate-contaminated groundwater. 

This study investigated the effect of several amendments on the degradation rate of perchlorate in an in situ environment.  A batch reactor study was conducted in laboratory microcosms using a control vs. lactate, polyvinyl alcohol (PVA), chitin and other amendments each containing 10 mg/L perchlorate and 1000mg/l amendment in replicate sets.  After 10 weeks, the results showed that PVA was the most effective in increasing the degradation rate of perchlorate. The results were below 1 mg/L.  Based on the batch study, a column study was conducted to evaluate the migration of polyvinyl alcohol (PVA) and degradation of perchlorate. 

Biological Treatment of Pentachlorophenol Contaminated Soils Using a Biopile System
Michael Snyder, P.E., Biogenie Corporation, 2085 Quaker Pointe Drive , Quakertown , PA   18951 Tel: 215-538-1729, Fax: 215-538 -8287, Email: msnyder@biogenie-env.com
LeeAnn Thomas, P.G.Canadian Pacific, 501 Marquette Ave. S. Suite 804 , Minneapolis , MN 55402 , Tel:  612-904-6130, Fax:  612-790-9498, Email: leeann_thomas@cpr.ca
Michael J. Borda, Ph.D., Golder Associates Inc., 200 Century Parkway, Mt. Laurel, NJ 08054
Tel:  856-793-2005, Fax:  856-793-2006, Email: mborda@golder.com

This poster documents the development of a Biopile system that was used for the successful biodegradation of 7200 cubic yards of pentachlorophenol (PCP) contaminated soils generated as a result of wood treatment operations.

Chemical analyses showed PCP concentrations in site soils ranging as high as 1400 mg/kg PCP.  Biological analyses of the soil revealed the presence of a viable microflora in sufficient concentrations to sustain a biological treatment process.  Laboratory treatability studies indicated that an 88% reduction of PCP concentration could be realized.  Results of the treatability study allowed the development of Biopile operating parameters designed to stimulate and sustain the biodegradation capacity of the indigenous microorganisms.  

Sample analysis conducted after 19 weeks of implementation indicated that over 58% of the Biopile had reached the primary cleanup goal of 120 mg/kg.  Sample analysis conducted during week 31 indicated an average PCP concentration of 11mg/kg, resulting in an average treatment efficiency of 93.6%.

Dehalococcoides Bioaugmentation Using Inter-Well Transfer of Bio-trap^® Samplers
Edward Sullivan, P.G., The Whitman Companies, Inc., 116 Tices Lane, Unit B-1, East Brunswick, NJ 08816, Tel: 732-390-5858, Fax: 732-390-9496, Email: esullivan@whitmanco.com
Greg Davis, Microbial Insights, Inc., 2340 Stock Creek Blvd., Rockford, TN  37853-3044, Tel: 865-573-8188, Fax: 865- 573-8133, Email: gdavis@microbe.com
Dora Ogles, Microbial Insights, Inc., 2340 Stock Creek Blvd., Rockford, TN  37853-3044, Tel: 865-573-8188, Fax: 865- 573-8133, Email: dogles@microbe.com
Keith McDermott, The Whitman Companies, Inc., 116 Tices Lane, Unit B-1, East Brunswick, NJ 08816, Tel: 732-390-5858, Fax: 732-390-9496, Email: kmcdermott@whitmanco.com

A pilot study was conducted at a site in New Jersey using a combination of nanoscale zero valent iron (nZVI) and emulsified soy oil to promote dual abiotic/biotic degradation of TCE in groundwater.  TCE concentrations in the injection area deep well (MW-17D) decreased by over one order of magnitude within six months of the injections.  Microbiological testing showed very high numbers of Dehalococcoides spp. microorganisms were present in ground water samples and in the biomass obtained from a Bio-trap^® sampler (4.45E+07 cells/bead) deployed in MW-17D.  In addition, Q-Expression (RNA) testing showed very high levels of the VC R-Dase functional gene (1.06E+07).  

A string of three (3) Bio-trap samplers supplied by Microbial Insights, Inc., were installed in MW-17D in November 2007.  One larger diameter Bio-Trap was designed to collect as much biomass as possible.  The two remaining Bio-Trap samplers were smaller diameter traps which were used for microbiological analysis.  The Bio-Trap samplers contain Bio-sep^® beads onto which microorganisms in the ground water will attach and colonize.  After approximately two months in MW-17D the string of Bio-Trap samplers were removed from the well.   One of the Bio-Traps was removed from the string and submitted to Microbial Insights for CENSUS^® analysis for Dehalococcoides.  The remaining two Bio-Traps were transferred into another site monitoring well (MW-18D) where less reductive dechlorination was evident.  MW-18D was also chosen because ground water geochemical conditions were similar to those in MW-17D.  Care was taken to minimize contact with atmospheric oxygen during the transfer process. 

Baseline sampling for Dehalococcoides and volatile organic compounds (VOCs) was conducted in MW-18D prior to Bio-Trap deployment in the well.   Subsequently, a post-deployment sampling program was implemented to evaluate whether the transferred microorganisms would proliferate in MW-18D and increase the rate of reductive dechlorination in that well.  The sampling program included the collection of ground water samples for Dehalococcoides and VOCs and analysis of the MW-18D Bio-Trap.