RESEARCH WITH MENTOR

THE EFFECTS OF CHROMIUM ON THE BIODEGRADATION OF GLUCOSE BY BACTERIA

 

Abstract: Our team was interested in studying the effects of Heavy Metals/ Chromium on the bacterial degradation of glucose.  Interests included the investigation of the link between chromium levels and nutrient concentrations. Studies found  that the rate of degradation was affected  not only by nutrient levels, but also by the concentration of chromium.

Introduction: Heavy metal contamination of soil represents one of the largest challenges for bioremediation today.  Studies have found that metals affect the growth, structure, and chemical activities of microorganisms.  Even though metals have an adverse affect on microorganisms, many have developed resistance strategies.  These microbes react differently to different metal types.  Understanding the reactions of these populations has become a main focus in this area of science.  Most of the experiments performed have been on Cadmium due to the abundance of it at Superfund sites.(1)  Mentor Dr. Derrick Brown had performed a preliminary experiment with cadmium.  Results showed that cadmium concentrations did affect bacterial degradation under various nutrient conditions. It was then suggested a different heavy metal be tried.  The USEPA considers all forms of chromium to be toxic although the level of toxicity along with the hazards and possible effects depends on the form the chromium is found in. Specific compounds appear to be toxic, while the actual element has a low toxicity rate.(2) The general population is exposed to chromium everyday when they eat, drink, and breathe.  The solubility and oxidation potential of the compounds is what dictates whether or not they will have a negative affect on humans. Typical careers that have a higher risk of chromium exposure are painters, copy machine workers, battery, candle, and dye, cement, and rubber makers.  Sources of chromium in the environment include landfill sites, industrial facilities, cooling towers, leather tanning, stainless steel, tobacco and busy roadways.  Little is known about the biological affect chromium has on the organisms in the environments where it is present, but chromium contamination of soils and groundwater is a widespread problem.(3)   For the reasons listed above the question arose; can bacteria react differently under various nutrient conditions and different chromium concentrations for our experiment.

Materials and Methods:

Method of measuring biodegradation:

 Biodegradation was measured using the HACH BOD Trak apparatus. This determined the Biochemical Oxygen Demand (BOD) by measuring a pressure drop in sealed bottles. The drop in pressure is due to bacterial utilization of available oxygen. HACH  BOD nutrient buffer pillows were used for the nutrient solution. The high nutrient solution was one pillow per bottle, the low nutrient solution was 1 mL of 2x HACH  BOD Solution ( 1 pillow in 150 mL deionized water) added to the sample bottle. Generic nutrient pillows were used.  Our glucose concentration was 300mg/L and we used Sphingomonas  paucimobilis as the bacterium.  The experiment was conducted in an incubator at 30 degrees Celsius for six days. 

Growth Substrate:     

Dextrose (D-Glucose) was the growth substrate. The degradation of glucose is represented by this formula, C6H12O6+6O2  yields 6CO2+6H2O.  The experimental range of the apparatus was determined to be 320 mg/L (O2).  We determined the range of the HACH apparatus to be 0-350mg/L.  To ensure that the lowest concentration of chromium used could easily be pipetted the sample volume was determined to be 160 mL. A Stock concentration of 4.8 g/L was used.  See Appendix 1

Chromium Volume:

Chromium Chloride (CrCl3+6H2O) has the formula weight of 266.45 g/mole.  Our Stock Chromium Solution needed a concentration of 0.16 moles/L, determined from previous experimentation. 4.263 g of CrCl3 will be added to 100mL of deionized water for Stock Chromium Solution.  See Appendix 2

Bacteria Preparation:

See Appendix 3

Experiment:

12 bottles were labeled 1-12. 1 and 2 being the controls, 3-7 being low nutrient, and 8-12 being high nutrient.  Each 160 mL sample consisted of the following:  10 mL dextrose (or DI for the control), 10 mL Bacteria Solution (or DI for the control), 0-5 mL Chromium Chloride Stock, and the remainder Deionized Water (DI).  

 

Data:

    CONTENTS OF BOTTLES USED IN EXPERIMENT  
   
  Sample Number Bacteria D/glucose Chromium Dionized Water Nutrient Levels
   
Controls 1 0 mL 10 mL 0 uL 150 mL High
  2 10 mL 0 mL 0 uL 150 mL High
   
Low Nutrient 3 10 mL 10 mL 0 uL 140 mL Low
  4 10 mL 10 mL 10 uL 140 mL Low
  5 10 mL 10 mL 100 uL 140 mL Low
  6 10 mL 10 mL 1 mL 139 mL Low
  7 10 mL 10 mL 5 mL 135 mL Low
   
High Nutrient 8 10 mL 10 mL 0 uL 140 mL High
  9 10 mL 10 mL 10 uL 140 mL High
  10 10 mL 10 mL 100 uL 140 mL High
  11 10 mL 10 mL 1 mL 139 mL High
  12 10 mL 10 mL 5 mL 135 mL High

Results and Discussion:

As seen from the data, the greater the chromium concentration, the greater the reduction of glucose biodegradation as measured in BOD (mg/L).   The graphed results indicate a direct relationship to the chromium concentrations over the seven day period for both set-ups. There is a more significant decrease in biodegradation of glucose in the high nutrient set-up.  The lower concentrations of chromium in the set-up does not show a large decrease in the biodegradation of glucose.   Notice on the high nutrient table .0mg/L and  .10mg/L are not present. The data collected showed no activity in these bottles.  This is due to experimental error in the apparatus or human error when preparing the samples.   Speculation was made that one of the bottles was not properly sealed and the other bottle was missing a solution.  Overall the data  indicates that there is a positive correlation between biodegradation and nutrient level.  (See Fig. 1 and 2)  Low nutrient levels in the bacterial solution tend to exacerbate the negative effect of chromium on BOD output.  Reasons for a nutritional effect range from putting the bacteria under stress without the added factor of increased levels of chromium, possible interference with enzyme action in the biodegradation pathway.

APPENDIX

Appendix 1

Growth Substrate:

            1mole dextrose (180.16 g/mole)+ 6moles oxygen (32g/mole)= 192g (O2) per mole Dextrose

            300mg/L Dextrose x 192 g O2/mole dextrose / 180.16 g dextrose/mole dextrose = 320 mg/L (O2)

                                                               BOD  = 320 mg/L

Appendix 2

Chromium Volume:

            (0.16 moles/L Stock Chromium Solution)(266.45 g/mole CrCl3 x 6H2O)(0.1 L DI)= 4.263g CrCl3

Appendix 3

Bacteria Preparation: 

1 July 2001 -  Plate S. paucilimobilis on 30 petri dishes of R2A agar.  Incubate at 30 degrees Celsius in Ziploc

                      bags (5 petri/bag)

5 July 2001 -  Harvest bacteria and suspend in 2x strength  HACH  BOD nutrient solution. 

                      Centrifuge 30 min. at 3000 RPM (-1500g)

                      Decant and resuspend in 2x strength HACH BOD nutrient solution.  Place on a magnetic stirrer at

                      room temperature.

6 July 2001 - Centrifuge bacteria 30 minutes at 3000 RPM.  Decant and resuspend in 150 mL DI with 1mL 2x

                      strength HACH BOD nutrient solution.

                       Spectrophotometer scan of bacteria from 200nm to 1000nm  A220= 1.498

                                                           Start experiment
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