Volume 1, Number 23

Remediation Weekly – Science & News Journal
www.RemediationWeekly.com
Volume 1, Number 23, November 15, 2004

Respirometry: A Breath of Fresh Air

By Chris Adams and Dr. Jan Czekajewski

Dr. Jan Czekajewski is president of Columbus Instruments, Columbus, Ohio.
Chris Adams is on staff with Columbus Instruments.
Phone 614-276-0861, Email: sales@colinst.com


The constant increase in pollution and other environmental issues has resulted in increased attention to control and remediation efforts. These efforts have resulted in an exhaustive catalog of cleanup techniques including respirometry. Over the past 20 plus years, “respirometry” has become synonymous with the Columbus Instruments Micro-Oxymax. It is used consistently as an easy, automated, low maintenance strategy for monitoring remediation in the research laboratory. Data acquired from the simple gas exchange of microorganisms (oxygen consumption and carbon dioxide production) has yielded valuable information on how nutrients are consumed and what concentrations or ratios impact growth activity. Since its inception, the Micro-Oxymax has found a vast range of applications. In addition to bioremediation and biodegradation, the accuracy and sensitivity of the Respirometer has attracted the attention of scientists who are applying it to new and exciting applications: new techniques in hydrocarbon cleanup, monitoring wastewater oxygen demand, monitoring hydrogen production in fermenting cultures, monitoring the oxidative photo-biodegradation of plastics, and cancerous tissue growth inhibitors are some of these new applications.

Background
In its most famous application, Dr. James Mueller (US EPA), used the Micro-Oxymax Respirometer (Figure 1) in the research effort to clean up the Exxon Valdez oil spill in Prince William Sound, Alaska. Samples were transported from Prince William Sound to the Gulf Breeze Research Laboratory in Gulf Breeze, Fla. Figure 2 shows some results of this experiment on oil contaminated sea grasses from Prince William Sound. E12V is a pure culture isolated from Prince William Sound. SBMIX is a partially defined mixed culture from Seal Beach, Calif. ESB and SBMIK-6 are pure cultures isolated from SBMIX. Results from the Micro-Oxymax suggested that SBMIX from Seal Beach was superior to other treatments; however, later stable isotopy analysis of the generated carbon dioxide showed that the carbon originated from the sea grasses rather than the oil. In the end, Dr. Mueller's research, which yielded a successful clean up of Prince William Sound, began the popular use of the Micro-Oxymax for biodegradation and bioremediation studies.

Bioremediation/Biodegradation
Further research in this area has resulted in additional treatment methods such as adding sewage sludge as an inoculum (1, 2) and using plant life for remediation stimulation (3). Under optimal environmental conditions, the indigenous microbes can effectively convert hydrocarbons to carbon dioxide. However, to maximize the efficiency of this process, precise amounts of supplements are added to the effected area. Specific concentrations of nutrients such as Nitrogen, Phosphorus, Potassium, etc., are necessary but not always sufficient; often times something more is needed to initiate or accelerate the bioremediation process. This “something” is dependant upon the environmental conditions and the heartiness of the naturally occurring microbes. The three research papers mentioned here describe this in more detail.

Wastewater Analysis
Besides bioremediation, the Micro-Oxymax has also been used in common wastewater analyses in both the laboratory and in the field. In a joint effort between Utrecht University and TNO Netherlands Institute of Applied Geosciences; the study examined the natural reduction capacity of aquifers. Increased pesticide use and farm land run-off has caused an excessive input of oxidants, resulting in the deterioration of groundwater quality (6, 10). Measuring the respiration of Bulk Organic Matter (BOM) in these aquifers allowed the continuous monitoring of the ongoing reduction-oxidation process, allowing early identification of contamination. Consequently, identified contaminated sites were then further tested, using respirometry, for suitability of in situ bioremediation techniques.

Bioleaching
Further uses for the Micro-Oxymax include bioleaching experiments. This is the process by which copper and other metals are released from ore or soil through a biological process utilizing microbes (7). In contrast to the heterotrophic bacteria in bioremediation and wastewater analysis, bioleaching utilizes autotrophic bacteria. These mineral oxidizing microbes consume both oxygen and carbon dioxide. The most effective way to measure carbon dioxide consumption is to measure the change in gas concentration directly. Monitoring this over time in a microbial sample requires great precision to yield useful data. The Micro-Oxymax, equipped with a highly sensitive infrared-based carbon dioxide gas analyzer, allows the measure of carbon dioxide consumption as well as oxygen consumption during the course of the experiment. The consumption rate of these gases directly correlate to the microbial activity and give a general status of the bioleaching process.

Photo-biodegradable Polymers
Environmentally friendly biodegradable plastics have been a hot topic for many years now. The sensitivity of the Micro-Oxymax Respirometer has allowed researchers to explore the fate of certain polymers after UV exposure (4, 5). Under an intensified UV light, specifically engineered polymers begin an oxidative process and begin to break down. The progress of this photo-oxidative process can be monitored by measuring the oxygen consumption rate; the higher the rate, the faster the biodegradable process.

Fermented Hydrogen Production
The superior sensitivity and accuracy of the Micro-Oxymax Respirometer has enabled research on hydrogen producing anaerobes for alternative fuel research. By adding a hydrogen sensor to the system, the amount of hydrogen produced by the microbes is measured directly (source protected as confidential). Pilot research has shown hydrogen production on the order of hundreds of milliliters per hour per liter of sample; full scale is projected to produce dramatically more.

Cancer Inhibitors
In the battle against cancer, researchers have used the Micro-Oxymax Respirometer to test cancer growth inhibitors by monitoring a reduction in respiration behavior of afflicted cell grown in vitro (8, 9) (additional sources protected as confidential). Cancerous tissue cultures have a significantly higher metabolic rate than do normal, healthy cultures. Monitoring the effects of Gossypol (Cotton seed extract) on prostate cancer cells has shown a dramatic decrease in oxygen consumption. This relates directly to the decrease in cancer-cell proliferation; the Gossypol is believed to disrupt oxidative phosphorylation process in the afflicted cells (further research continues).

The applications listed above represent only a few examples of how the Micro-Oxymax is used today. Increased work in bioremediation efforts world wide has brought respirometry into the spotlight. But its capabilities have attracted scientists from many different backgrounds for a myriad of applications. Undoubtedly, the Respirometer will find a home in an increasing number of research laboratories.

Bibiography

1) Biodegradation of Petroleum Hydrocarbons in Landfarm Soil I) Effect of Nutrient Amendment
R.B. Chen, T.M. AL-Ibrahim, H.H. Chen, and W.J. Gwathney 1st Specialty Conference on Env. Issues, 12/4-6/95

2) Biodegradation of Petroleum Hydrocarbons in Landfarm Soil II) Use of Sewage Sludge as Amendment in Soil
R.B. Chen, T.M. AL-Ibrahim, H.H. Chen, and W.J. Gwathney 1st Specialty Conference on Env. Issues, 12/4-6/95

3) Trees Stimulate Remediation at Fuel Oil Contaminated Site
Eric P Carman, Tom L Crossman, & Edward G Gatliff Soil & Groundwater Cleanup, Feb/Mar 40-44 (1998).

4) Photo-Oxidative degradation of poly(2,6-dimethyl-1, 4-phenylene oxide) in the presence of concentrated hydroxy peroxide: the role of hydroxy (HO.) and hydroperoxy (HO2) radicals
H. Kaczmarek, L.A. Linden, J.F. Rabek Polymer Degradation and Stability 47(1995) pp. 175-188.

5) Application of a new generation of computerized apparatus for the study of oxygen uptake and production of CO and CO2 during photo-(thermal) oxidation of polymers
J. Czekajewski, L. Nennerfelt, H. Kaczmarek, and J.F. Rabek Acta Polymer, 45, 369-374 (1994).

6) Determining the Reactivity of Reduced Components in Dutch Aquifer Sediments
Niels hartog, jasper griffioen, Pim F. Van Bergen And Cornelis H. Van Der Weijden Impact of Human Activity on Groundwater Dynamics (Proceedings of a Symposium Held During the Sixth IAHS Scientific Assembly at Maastricht, The Netherlands, July 2001). IAHS Publ. No. 269 pp.221-227

7) Development of Respirometry Methods to Assess the Microbial Activity of Thermophilic Bioleaching Archaea
Chris A. du Pleiss, Paul Barnard, Kurt Naldrett, Sanet H. de Kock Journal of Microbiological Methods 47 (2001) 189-198

8) Use of Computerized O2/CO2 Respirometer in Monitoring Respiration of Prostate Cancer Tissue
Ghosh PK, Jiang D, Lin Y, & Czekajewski J Abstract, Society for the Study of Reproduction Meeting 1998

9) Effects of Gossypol on O2 Consumption and CO2 Production in Human Prostate Cancer Cells
Jiahua Jiang, Pradip Ghosh, Samuel K. Kulp, Yasuro Sugimoto, Suling Liu, Jan Czekajewski, Hsiang-Lin Chang, and Young C. Lin Anticancer Research 22: 1491-1496 (2002)

10) Respiration of Bulk Organic Matter in aquifer sediments with the Micro Oxymax
N. Hartog, B. van der Grift, J. Griffioen, and H. van Liere Mineralogical Magazine, 1998, Volume 62A, pp. 579-580.

Further Reading

Bioaugmentation and Biostimulation: A Paradox Between Laboratory and Field Results
K. Lee, G.H. Tremblay, J. Gauthier, S.E. Cobanli, M. Griffin 1997 Oil Spill Conference (American Petroleum Institute pub. no. 4651) pp. 697-705.

Current research in Micro Oxymax
William A. Hopkins Savannah River Ecology Laboratory 8/21/99

Full List of research papers available at www.respirometer.com; see Micro-Oxymax references section.