This study developed a new most probable number (MPN) method to enumerate hydrocarbon-degrading bacteria using 96-well microtiter plates, diesel as the sole carbon source, and resazurin as the indicator. Testing with pure cultures of Pseudomonas putida and Escherichia coli showed similar bacterial counts between the new MPN method and plate counts, and the MPN method selectively detected only P. putida. Environmental samples of compost and contaminated soil also yielded expected results, detecting low bacterial counts in compost and high counts adapted to hydrocarbons in soil. The new MPN method provides an effective way to enumerate hydrocarbon-degrading bacteria relevant to bioremediation of contaminated sites.

1. ENUMERATION OF HYDROCARBON-DEGRADING BACTERIA BY MOST
PROBABLE NUMBER USING RESAZURIN AS INDICATOR
Jubany I.1, Calderer M. 1, González C. 1, Corcho D. 2, Muelle A. 2 ,Martí V.1,3
1CTM, Technological Center Foundation, Environmental Technology Area (UPC), Av. Bases de Manresa, 1, E-08242 Manresa (Spain)
2 INTRAVAL S.L. Llull, No. 109, 2nd floor, E-08005 Barcelona (Spain)
3Department of Chemical Engineering, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal, 647, E-08028, Barcelona (Spain)
INTRODUCTION
Enumeration of bacteria is very useful in the biological soil treatment in order to detect and quantify the
microorganisms involved in the bioremediation. One of the most widely used methods is the most
probable number (MPN) technique. A new MPN method for enumeration of hydrocarbon-degrading
bacteria (HDB) was developed in this work using 96-wells microtiter plates for incubation, commercial
Figure 1. Chemical change of resazurin and
diesel as the carbon source and the use of resazurin as activity indicator. The method was a modification resulting colour change.
of the work by Wren and Venosa (1996).
Resazurin is an oxazine which upon reduction changes colour from blue to pink due to the loss of an oxygen atom loosely bound (see Figure 1).
MATERIALS AND METHODS
Developed method: MPN with resazurin and diesel Bacteria enumeration using plate count:
For each sample, tenfold serial dilutions were performed in a saline Plate count was performed as an indicator of the total
buffer solution (8.5 g/L NaCl). 96-wells microtiter plates were heterotrophic bacteria as described in APHA (1998). Two
inoculated by adding 20 μL of each dilution (8 wells per dilution) to different growth substrates were used. A nutrient broth was
180 μL of mineral medium (2.0 g/L NH4Cl, 0.89 g/L KH2PO4, 1.25 g/L used for pure cultures enumeration and R2A Agar was used
Na2HPO4, 0.6 mg/L FeCl3). 10 μL of diesel were added to each well as for environmental samples. Plates were incubated for 7 days
a sole substrate. Each sample was tested in duplicate. Plates were at 28 ºC. After this period, only plates having 30 to 300
incubated at 25 ºC for 21 days and then, 50 μL of resazurin (4.5 mg/L) colonies were considered for couting. All samples were
were added to each well. Positive wells (pink coloured) were scored tested in duplicate
after 48 h. The MPN was calculated.
Pure cultures Environmental samples
Tested
samples: Pseudomonas putida Escherichia coli Compost from municipal wastewater Soil from hydrocarbon-
(CECT 845) (CECT 101) treatment sludge contaminated site
HDB No HDB Low HDB content High HDB content
RESULTS
Table 1. Results obtained with pure cultures (p=0.5) in cells/g (dry matter) • Same count of bacteria with plate count and MPN with the nutrient
P. putida E.coli broth were obtained
Method Medium Count Min Max Count Min Max • The same count of bacteria for P. putida was obtained with the
Plate count Nutrient broth 1.75·108 1.67·108 1.82·108 2.74·108 2.23·108 3.25·108 developed method and the plate count.
Nutrient broth 2.60·108 1.01·108 6.69·108 6.21·108 2.42·108 1.60·109 • No E. coli was detected with the developed method (no false
MPN
Diesel 1.22·108 4.74·107 3.13·108 Not detected positive responses were detected)
Table 2. Results obtained with environmental samples (p=0.5) in cells/g (dry matter) • Low amount of HDB in the compost
Compost sample Hydrocarbon-contaminated soil sample was detected as expected.
Method Medium Count Min Max Count Min Max • Similar count of bacteria in the
Plate count R2A Agar 1.10·1010 9.10·109 1.09·1010 6.60·107 2.590·107 7.30·107 hydrocarbon-contaminated soil was Figure 2. MPN in 96-well microtiter plates.
MPN Diesel 1.57·103 6.11·102 7.03·103 2.59·107 1.01·107 6.65·107 A. P. putida in nutrient broth. B. P. putida in
obtained with both methods, diesel medium and resazurin. C. E. coli in
indicating that all bacteria are HDB. diesel medium and resazurin
CONCLUSIONS
The developed MPN method was based on three aspects: (1) the use of 96-wells microtiter plates for incubation, (2) the use of commercial diesel as
the carbon source, and (3) the use of resazurin as activity indicator.
The method resulted to be appropriate when testing with pure cultures since bacteria counts where comparable with counts obtained with the
plate count method. It was confirmed that resazurin did not react with diesel which could have caused false positives.
 Very different HDB was found in compost and contaminated soil samples indicating that the bacteria in the contaminated soil were already adapted
to hydrocarbons.
 These results demonstrated that the developed method could be used for the enumeration of hydrocarbon-degrading bacteria in the field of
biological treatment of hydrocarbon-contaminated soil where, usually, amendments based on compost are used.
REFERENCES
• Wrenn , B.A. and Venosa A.D. (1996). Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by most-probable-number procedure. Canadian Journal of Microbiology, 42(3), 252-258
• APHA (1998). Standard methods for the examination of water and wastewater. Washington: American Public Health Association.