CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia -LRB- AML -RRB- of older age Clinical resistance to chemotherapy in acute myeloid leukemia -LRB- AML -RRB- is associated with the expression of the multidrug resistance -LRB- MDR -RRB- proteins P-glycoprotein , encoded by the MDR1/ABCB1 gene , multidrug resistant-related protein -LRB- MRP/ABCC1 -RRB- , the lung resistance-related protein -LRB- LRP -RRB- , or major vault protein -LRB- MVP -RRB- , and the breast cancer resistance protein -LRB- BCRP/ABCG2 -RRB- . The clinical value of MDR1 , MRP1 , LRP/MVP , and BCRP messenger RNA -LRB- mRNA -RRB- expression was prospectively studied in 154 newly diagnosed AML patients ≥ 60 years who were treated in a multicenter , randomized phase 3 trial . Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count -LRB- respectively , p < 0.05 , p < 0.001 , p < 0.001 and p < 0.001 -RRB- . Higher BCRP mRNA was associated with secondary AML -LRB- p < 0.05 -RRB- . MDR1 and BCRP mRNA were highly significantly associated -LRB- p < 0.001 -RRB- , as were MRP1 and LRP mRNA -LRB- p < 0.001 -RRB- expression . Univariate regression analyses revealed that CD34 expression , increasing MDR1 mRNA as well as MDR1/BCRP coexpression , were associated with a lower complete response -LRB- CR -RRB- rate and with worse event-free survival and overall survival . When adjusted for other prognostic actors , only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate -LRB- p = 0.03 -RRB- , thereby identifying a clinically resistant subgroup of elderly AML patients . Introduction Clinical resistance to chemotherapy in acute myeloid leukemia -LRB- AML -RRB- is often associated with the expression of -LRB- membrane -RRB- transport-associated multidrug resistance -LRB- MDR -RRB- proteins . Expression of P-glycoprotein -LRB- P-gp -RRB- , encoded by the MDR1 gene , is an independent adverse prognostic factor for response and survival in de novo AML -LSB- 1 -- 4 -RSB- . Moreover , it has been shown that besides P-gp , also the MDR-related protein -LRB- MRP1/ABCC1 -RRB- and the lung resistance-related protein -LRB- LRP -RRB- , also designated as the major vault protein -LRB- MVP -RRB- , are expressed in AML . However , the prognostic significance of the latter resistance proteins has not been settled -LSB- 3 , 5 -- 7 -RSB- . Some years ago , a new drug resistant protein , i.e. , the breast cancer resistance protein -LRB- BCRP/ABCG2 -RRB- , which is the equivalent of the mitoxantrone -LRB- MXT -RRB- resistant protein and the placental ABC transporter -LRB- ABCP -RRB- , was found to be expressed in AML -LSB- 8 -- 13 -RSB- . The precise role of either resistance proteins among poor risk AML such as in patients of older age has not been established . This study prospectively investigated the relevance of MDR1 , MRP1 , LRP , and BCRP messenger RNA -LRB- mRNA -RRB- expression in combination with known prognostic characteristics like CD34 expression , white blood cell -LRB- WBC -RRB- count , and secondary AML as possible denominators of response and survival in patients with AML aged 60 + who were treated in the same clinical trial . Patients and methods Patients A group of 154 patients with AML aged 60 years or older were included in the present study . All patients were enrolled between May 1997 and February 1999 in an international , multigroup , randomized phase 3 trial performed under auspices of the Dutch -- Belgian Hemato-Oncology Cooperative Group and the UK Medical Research Council -LSB- 14 -RSB- . In that trial , 419 eligible white patients ≥ 60 years with previously untreated de novo and secondary AML -LRB- M0 -- M2 and M4 -- M7 according to the French -- American -- British -LSB- FAB -RSB- classification -LSB- 15 -RSB- -RRB- were randomized to receive two cycles of induction chemotherapy consisting of daunorubicin -LRB- DNR -RRB- and cytarabine -LRB- ara-C -RRB- with or without the P-gp inhibitor PSC-833 -LRB- Valspodar , Amdray ® ; Novartis Pharma , Basle , Switzerland -RRB- . Patients in both arms in complete remission after these two cycles were to receive one consolidation consisting of ara-C , MXT , and etoposide . Inclusion criteria , clinical characteristics , treatment , and outcome of the phase 3 trial have been previously reported -LSB- 14 -RSB- . Bone marrow -LRB- BM -RRB- aspirates had been collected at diagnosis for the analysis of P-gp function and expression , as described previously -LSB- 14 -RSB- . Selection of patients for our study was based on availability of sufficient purified AML blast samples in our tissue bank , which was the case for 154 patients . This study was approved by the ethics committees of the participating institutions and was conducted in accordance with the Declaration of Helsinki . Written informed consent was obtained from all patients before randomization . Methods BM aspirates were obtained in heparinized tubes . Mononuclear BM cells were collected by Ficoll Hypaque density gradient centrifugation -LRB- density 1.077 g/m3 ; Pharmacia , Uppsala , Sweden -RRB- . To obtain purified samples with more than 85 % of blasts , T-cell depletion and adherence depletion was performed as previously described -LSB- 16 -RSB- . Cells were cryopreserved in Dulbecco modified Eagle medium -LRB- DMEM ; Gibco , Paisley , UK -RRB- supplemented with 10 % dimethyl sulfoxide -LRB- Merck , Darmstadt , Germany -RRB- and 20 % fetal calf serum -LRB- FCS ; Gibco -RRB- and stored in liquid nitrogen . On the day of the experiments , BM cells were thawed . Cells were washed and resuspended in DMEM supplemented with 10 % FCS . Before RNA and DNA isolation , cells were washed with phosphate-buffered saline -LRB- Gibco -RRB- . MDR1 , MRP1 , LRP , and BCRP mRNA analysis The drug resistance proteins were analyzed using the methods that we previously reported -LSB- 11 -RSB- . In brief , total RNA was isolated using the TRISOLV ™ extraction as described by the manufacturer -LRB- Biotecx , Houston , TX -RRB- . RNA was aliquoted and stored at − 80 °C . RNA samples were analyzed for RNA integrity by gel electrophoresis . cDNA was synthesized by the use of the TaqMan Reverse Transcription Reagents -LRB- Applied Biosystems , Foster City , CA -RRB- , diluted , aliquoted , and stored at − 80 °C . Quantitative RT-PCR was used to measure the mRNA expression levels of MDR1 , MRP1 , LRP , and BCRP by Taqman-chemistry on an ABI PRISM 7700 sequence detector -LRB- Applied Biosystems -RRB- using two endogenous reference genes , i.e. , glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase . Definition of endpoints The clinical endpoints have been defined previously -LSB- 14 -RSB- . In brief , complete response -LRB- CR -RRB- was defined as a normocellular BM with < 5 % blasts , no Auer rods , and no evidence of extramedullary involvement . Because data on peripheral blood recovery within 60 days were not always available , they were not considered as a criterium for CR . Patients who relapsed or died within 28 days after CR were considered as not having achieved a CR . Event-free survival -LRB- EFS -RRB- was calculated from the date of randomization until no CR on induction therapy , relapse after CR , or death in CR , whichever came first . Patients who did not reach CR were considered failure for EFS at 1 day after randomization . Disease-free survival -LRB- DFS -RRB- was determined for all patients who achieved CR on induction therapy and was calculated from the date of CR until relapse or death , whichever came first . Overall survival -LRB- OS -RRB- was measured from randomization until death from any cause . Patients who were still alive at the date of last contact were then censored . Statistical analysis The original phase 3 trial had been designed to detect with a power of 80 % an increase in 2-year EFS from 9.5 % in the control arm -LRB- without PSC-833 -RRB- to 18 % in the PSC-833 arm -LRB- two-sided significance level α = 0.05 -RRB- and included 419 eligible patients . mRNA data were obtained from a subset of 154 patients with sufficient BM samples in our tissue bank available for analysis . Baseline parameters of interest were MDR1 , MRP1 , LRP , and BCRP mRNA expression . Clinical endpoints were CR rate , EFS , DFS , and OS . Baseline characteristics of patients with or without mRNA expression data available were compared using the Fisher exact test or the Pearson χ2 test in case of discrete variables , whichever appropriate , or the Wilcoxon rank-sum test in case of continuous variables . The association between patient baseline characteristics and mRNA expression levels was analyzed using the Pearson χ2 test of the Spearman rank correlation test , whichever was appropriate . The prognostic value of mRNA levels with respect to CR rate was determined using logistic regression -LSB- 17 -RSB- whereas the impact of MDR1 , MRP1 , LRP , and BCRP on EFS , DFS , and OS was analyzed with Cox regression analysis -LSB- 18 -RSB- . For this purpose , the natural logarithm of the mRNA expression levels of the four resistance genes were included in the analyses because of the very skewed distribution of the original mRNA levels . In addition , the outcome of patients with coexpression of MDR1 and BCRP was evaluated to confirm the poor prognosis of AML with MDR1/BCRP coexpression reported by Benderra et al. -LSB- 19 -RSB- in patients with a median age of 45 years or older . These patients were defined as having mRNA levels of these two drug resistance genes equal to or higher than the median . Their outcome was compared to the other patients with at least one of the MDR1 and BCRP mRNA expression levels below the median . Logistic regression and Cox regression analyses were performed unadjusted , as well as adjusted for other prognostic factors , i.e. , secondary AML , natural logarithm of WBC count , square root of percentage CD34 + cells , and cytogenetic risk -LRB- favorable/intermediate versus unfavorable versus unknown -RRB- , as well as for treatment arm in the phase 3 trial , as about half of the patients had been randomized to receive PSC-833 in addition to their chemotherapy . Kaplan -- Meier curves -LSB- 20 -RSB- were generated to illustrate survival and were compared using the log-rank test -LSB- 21 -RSB- . All reported p values are two-sided and , in view of the exploratory nature of these analyses , were calculated without adjustment for multiple testing . p values ≤ 0.05 were considered statistically significant . Results In the phase 3 trial , a total of 419 untreated patients with AML aged 60 years and older were randomized to receive two induction cycles with or without PSC-833 . As reported , no difference was found between both treatment arms as regards CR rate -LRB- 54 % in the PSC-833 arm versus 48 % in the control arm , p = 0.22 -RRB- , 5-year EFS -LRB- 7 versus 8 % , p = 0.53 -RRB- nor DFS -LRB- 13 versus 17 % , p = 0.06 -RRB- and OS -LRB- 10 % in both arms , p = 0.52 -RRB- -LSB- 14 -RSB- . We previously reported the role of functional MDR1 expression with respect to clinical outcome in these patients . In 154/419 of the patients , sufficient BM cells were available in our tissue bank to investigate the mRNA expression level of the drug resistance genes MDR1 , MRP1 , LRP , and BCRP . This subgroup consisted of a representative group according to age , gender , CD34 expression , cytogenetics , and FAB classification -LRB- Table 1 -RRB- . In this test group , a higher WBC count at diagnosis was observed than in the other 265 patients , and relatively more patients had been randomized to the PSC-833 arm -LRB- 57 versus 45 % , p = 0.02 -RRB- . There was no significant difference in the levels of MDR1 , MRP1 , LRP , nor BCRP mRNA expression between the two treatment arms -LRB- data not shown -RRB- . The CR rate and survival endpoints were also similar in both patient groups -LRB- Table 1 -RRB- . However , patients with mRNA data in the PSC-833 arm had a higher CR rate -LRB- 61 versus 40 % , p = 0.02 -RRB- , whereas this was 54 versus 48 % -LRB- p = 0.22 -RRB- in all 419 patients . Table 1Comparison between patients with or without data available for expression of the drug resistance genes Drug resistance genes evaluated Yes N -LRB- % -RRB- No N -LRB- % -RRB- Total N -LRB- % -RRB- pNumber of patients154265419Patient characteristicsMedian age , -LRB- range -RRB- 67 -LRB- 60 -- 85 -RRB- 68 -LRB- 58 -- 85 -RRB- 67 -LRB- 58 -- 85 -RRB- 0.52 Sex0 .26 Male86 -LRB- 56 -RRB- 163 -LRB- 62 -RRB- 249 -LRB- 59 -RRB- Female68 -LRB- 44 -RRB- 102 -LRB- 38 -RRB- 170 -LRB- 41 -RRB- Secondary AML31 -LRB- 20 -RRB- 73 -LRB- 28 -RRB- 104 -LRB- 25 -RRB- 0.09 Median WBC count -LRB- × 109/l ; range -RRB- 19.1 -LRB- 0.1 -- 389 -RRB- 5.6 -LRB- 0.5 -- 300 -RRB- 8.9 -LRB- 0.1 -- 389 -RRB- 0.001 N146243389Median % CD34 + , -LRB- range -RRB- 32.5 -LRB- 0.1 -- 97.9 -RRB- 29.7 -LRB- 0.1 -- 93.7 -RRB- 30.3 -LRB- 0.1 -- 97.9 -RRB- 0.50 N152157309Cytogenetic risk classificationa0 .12 Favorable3 -LRB- 3 -RRB- 2 -LRB- 1 -RRB- 5 -LRB- 2 -RRB- Intermediate90 -LRB- 80 -RRB- 132 -LRB- 73 -RRB- 222 -LRB- 76 -RRB- Unfavorable19 -LRB- 17 -RRB- 47 -LRB- 26 -RRB- 66 -LRB- 23 -RRB- No data42 -LRB- n.i. -RRB- 84 -LRB- n.i. -RRB- 126 -LRB- n.i. -RRB- Treatment arm randomized0 .02 DNR/ara-C 66 -LRB- 43 -RRB- 145 -LRB- 55 -RRB- 211 -LRB- 50 -RRB- DNR/ara-C + PSC-83388 -LRB- 57 -RRB- 120 -LRB- 45 -RRB- 208 -LRB- 50 -RRB- Treatment outcomeCR rate , % -LRB- 95 % CI -RRB- 52 -LRB- 44 -- 60 -RRB- 50 -LRB- 44 -- 56 -RRB- 51 -LRB- 46 -- 56 -RRB- 0.73 EFS , % -LRB- 95 % CI -RRB- 0.72 1 year23 -LRB- 17 -- 30 -RRB- 23 -LRB- 18 -- 28 -RRB- 23 -LRB- 19 -- 27 -RRB- 5 years9 -LRB- 5 -- 14 -RRB- 7 -LRB- 4 -- 11 -RRB- 8 -LRB- 5 -- 11 -RRB- DFS , % -LRB- 95 % CI -RRB- 0.81 1 year38 -LRB- 27 -- 48 -RRB- 39 -LRB- 31 -- 48 -RRB- 39 -LRB- 32 -- 45 -RRB- 5 years17 -LRB- 10 -- 26 -RRB- 14 -LRB- 9 -- 21 -RRB- 15 -LRB- 11 -- 21 -RRB- OS , % -LRB- 95 % CI -RRB- 0.31 1 year42 -LRB- 34 -- 50 -RRB- 41 -LRB- 35 -- 46 -RRB- 41 -LRB- 36 -- 46 -RRB- 5 years14 -LRB- 9 -- 20 -RRB- 8 -LRB- 5 -- 12 -RRB- 10 -LRB- 7 -- 14 -RRB- The results indicate that , apart from WBC count , there are no differences between the two subgroups.N number of patients with data -LRB- if not available for all patients -RRB- ; n.i. not included when calculating percentagesaClassification of cytogenetic abnormalities only for 293 patients with successful cytogenetics . Favorable risk : t -LRB- 8 ; 21 -RRB- , inv -LRB- 16 -RRB- or t -LRB- 16 ; 16 -RRB- . Unfavorable risk : the presence of monosomies or deletions of chromosomes 5 or 7 , abnormalities of the long arm of chromosome 3 -LRB- q21 ; q26 -RRB- , t -LRB- 6 ; 9 -RRB- , abnormalities involving the long arm of chromosome 11 -LRB- 11q23 -RRB- , or complex cytogenetic abnormalities -LRB- defined as at least three unrelated cytogenetic abnormalities in one clone -RRB- . Patients who did not meet the criteria for favorable or unfavorable risk were classified as intermediate risk -LSB- 14 -RSB- . The mRNA expression levels of the resistance genes were not significantly associated with the age of the patients -LRB- Table 2 -RRB- . MRP1 and LRP expression showed a strong positive correlation with WBC count . Negative associations of MDR1 and BCRP with WBC count were observed . A significant positive association was found between CD34 and MDR1 and also with BCRP mRNA expression . No significant correlation was found between MRP1 nor LRP , and CD34 expression -LRB- Table 2 -RRB- . Interestingly , secondary AML cases had a significantly higher expression of BCRP -LRB- p < 0.05 -RRB- and lower MRP1 and LRP levels -LRB- both p < 0.01 , Table 2 -RRB- . In the vast majority of our patients , also P-gp efflux and expression data were available . Function and expression data and MDR1 mRNA expression levels were highly correlated -LRB- p < 0.001 -RRB- , which was published recently -LSB- 22 -RSB- . Table 2Association between clinical patient characteristics and the mRNA expression of the four drug resistance genes and MDR1/BCRP coexpression mRNA expression ofMDR1MRP1LRPBCRPMDR1/BCRP coexpressionCharacteristic Age0 .15 − 0.01 − 0.090.090.07 -LRB- 153 -RRB- -LRB- 153 -RRB- -LRB- 153 -RRB- -LRB- 137 -RRB- -LRB- 147 -RRB- Secondary AML0 .06 − 0.22 ** − 0.21 ** 0.19 * 0.12 -LRB- 153 -RRB- -LRB- 153 -RRB- -LRB- 153 -RRB- -LRB- 137 -RRB- -LRB- 147 -RRB- WBC count − 0.17 * 0.28 *** 0.36 *** − 0.36 *** − 0.35 *** -LRB- 145 -RRB- -LRB- 145 -RRB- -LRB- 145 -RRB- -LRB- 131 -RRB- -LRB- 139 -RRB- CD34 +0.54 *** 0.14 − 0.080.17 * 0.27 ** -LRB- 151 -RRB- -LRB- 151 -RRB- -LRB- 151 -RRB- -LRB- 135 -RRB- -LRB- 145 -RRB- Unfavorable 0.11 − 0.05 − 0.23 * 0.130.10 Cytogenetic risk -LRB- 111 -RRB- -LRB- 111 -RRB- -LRB- 111 -RRB- -LRB- 98 -RRB- -LRB- 106 -RRB- Unfavorable cytogenetic risk was defined by the presence of monosomies or deletions of chromosomes 5 or 7 , abnormalities of the long arm of chromosome 3 -LRB- q21 ; q6 -RRB- , t -LRB- 6 ; 9 -RRB- , abnormalities involving the long arm of chromosome 11 -LRB- 11q23 -RRB- , or complex cytogenetic abnormalities -LRB- defined as at least three unrelated cytogenetic abnormalities in one clone -RRB- Each cell displays the Spearman rank correlation coefficient between two variables and , between brackets , the number of patients with both variables available . * p < 0.05 ; ** p < 0.01 ; *** p < 0.001 In this cohort of patients of higher age with AML , MDR1 and BCRP were highly associated -LRB- p < 0.001 -RRB- , just as were MRP1 and LRP mRNA -LRB- p < 0.001 ; Fig. 1 -RRB- . A negative association was found between BCRP and MRP1 and between BCRP and LRP -LRB- both p < 0.001 ; Fig. 1 -RRB- . The 40 patients with coexpression of BCRP and MDR1 had significantly higher CD34 expression -LRB- median 39.5 % -LSB- range 0.1 -- 97.7 % -RSB- versus 25.9 % -LSB- range 0.1 -- 97.9 % -RSB- ; p = 0.001 -RRB- and a lower WBC count -LRB- median 4.5 -LSB- range 0.8 -- 300 -RSB- × 109/l versus 28.1 -LSB- range 0.1 -- 389 -RSB- × 109/l ; p < 0.001 -RRB- . No significant correlation of MDR1 , BCRP , or coexpression of MDR1 and BCRP was found with unfavorable cytogenetics -LRB- p = 0.4 ; Table 2 -RRB- . Fig. 1Association between MDR1 , MRP1 , LRP , and BCRP mRNA expression levels . Each dot represents the expression of two drug resistance genes in one patient . The Spearman rank correlation coefficient has been calculated , along with the corresponding p value . Both the x - and y-axis have a logarithmic scale trim -LRB- X -RRB- * indicates that the 2.5 % smallest and largest values of X have been shrinked ; r , Spearman rank correlation coefficient ; and p , p value . The results show a significant positive correlation between MDR1 and BCRP mRNA expression as illustrated by the p value and correlation coefficient . In addition , MRP1 and LRP are highly associated . BCRP shows a negative correlation with MRP1 and LRP To assess the clinical relevance of the four resistance genes , their expression was evaluated with regard to CR rate and survival data , respectively . The median follow-up of the 25 patients still alive was 58 months -LRB- range , 1 -- 80 months -RRB- . Univariate logistic regression analysis showed that higher MDR1 mRNA expression predicted for a lower CR rate -LRB- log -LSB- MDR1 -RSB- : odds ratio -LSB- OR -RSB- = 0.75 , 95 % confidence interval -LSB- CI -RSB- 0.61 -- 0.93 , p = 0.009 -RRB- , whereas MRP1 , LRP , and BCRP mRNA were not associated with CR -LRB- Fig. 1 -RRB- . MDR1 expression was also associated with a worse EFS -LRB- log -LSB- MDR1 -RSB- : hazard ratio -LSB- HR -RSB- = 1.14 , 95 % CI 1.03 -- 1.27 , p = 0.01 -RRB- and OS -LRB- log -LSB- MDR1 -RSB- : HR = 1.16 , 95 % CI 1.05 -- 1.29 , p = 0.004 -RRB- . Similar results were also obtained for MDR1/BCRP coexpression -LRB- Table 3 ; Fig. 2 -RRB- . When the analyses were performed with adjustment for other prognostic factors , as described in the `` Statistical analysis '' , only MDR1/BCRP mRNA coexpression remained significantly associated with a lower CR rate -LRB- OR = 0.37 , 95 % CI 0.15 -- 0.91 , p = 0.03 -RRB- , whereas a trend was observed for worse EFS -LRB- Table 3 -RRB- . On the other hand , higher CD34 expression was significantly associated with a lower CR rate -LRB- square root -LSB- CD34 -RSB- : OR = 0.86 , 95 % CI 0.76 -- 0.98 , p = 0.02 -RRB- and with worse EFS -LRB- HR = 1.12 , 95 % CI 1.06 -- 1.19 , p < 0.001 -RRB- , DFS -LRB- HR = 1.19 , 95 % CI 1.09 -- 1.30 , p < 0.001 -RRB- , and OS -LRB- HR = 1.17 , 95 % CI = 1.10 -- 1.25 , p < 0.001 -RRB- . Table 3Prognostic value of drug resistance gene expression w.r.t. CR rate , EFS , DFS from CR , and OS CR rateEFSDFSOSOR95 % CIpHR95 % CIpHR95 % CIpHR95 % CIpMDR1 Univariate0 .750.61 -- 0.930.0091.141.03 -- 1.270.011.130.97 -- 1.300.111.161.05 -- 1.290.004 Adjusted0 .770.58 -- 1.030.081.050.91 -- 1.210.480.950.77 -- 1.180.671.000.87 -- 1.160.97 MRP1 Univariate1 .060.83 -- 1.350.631.020.90 -- 1.150.791.070.89 -- 1.290.471.110.97 -- 1.260.12 Adjusted1 .220.90 -- 1.660.201.000.87 -- 1.150.981.120.92 -- 1.370.261.050.91 -- 1.210.54 LRP Univariate1 .160.94 -- 1.430.160.950.86 -- 1.060.360.980.84 -- 1.140.790.970.87 -- 1.080.60 Adjusted1 .220.93 -- 1.610.150.990.87 -- 1.120.831.060.89 -- 1.270.520.980.86 -- 1.120.78 BCRP Univariate0 .840.66 -- 1.060.141.040.91 -- 1.180.580.950.77 -- 1.160.600.960.84 -- 1.100.58 Adjusted0 .790.59 -- 1.06 0.120.990.86 -- 1.140.920.840.66 -- 1.060.140.900.77 -- 1.050.19 MDR1/BCRP co-expression Univariate0 .380.18 -- 0.800.011.631.11 -- 2.370.011.650.90 -- 3.010.111.471.00 -- 2.160.05 Adjusted0 .370.15 -- 0.92 0.031.530.98 -- 2.380.061.370.67 -- 2.820.391.160.74 -- 1.830.51 Results of logistic -LRB- for CR rate -RRB- and Cox regression -LRB- for survival -RRB- analyses , either univariate -LRB- = unadjusted -RRB- or adjusted for treatment arm , secondary AML , WBC count -LRB- natural logarithm -RRB- , % CD34 + -LRB- square root -RRB- , and cytogenetic risk -LRB- favorable/intermediate versus unfavorable versus unknown -RRB- , are shown for each of the four drug resistance genes MDR1 , MRP1 , LRP , and BCRP -LRB- natural logarithm of mRNA expression levels -RRB- and for MDR1/BCRP co-expression . Fig. 2Survival of elderly AML patients with and without coexpression of MDR1 and BCRP mRNA . a Event-free survival , b disease-free survival , c overall survival . pos indicates patients with coexpression of MDR1 and BCRP ; and other , patients without coexpression Discussion This is the first comprehensive analysis of the effect of the major classical MDR genes in a cohort of elderly patients with AML homogeneously treated in a prospective clinical trial -LSB- 14 -RSB- . A wide range of expression of the various resistance genes was observed , consistent with previous studies and with comparable median values -LSB- 9 -- 11 , 23 -RSB- . Our results show that MRP1 , LRP , and BCRP are not associated with CR rate or survival endpoints in patients with AML aged 60 years or older , indicating that the clinical relevance of the expression of these genes is limited in this patient population . This study confirms previous reports , which showed the unique prognostic role of MDR1 expression -- which was however highly correlated with CD34 expression -- in drug resistance in elderly AML -LRB- Table 3 -RRB- , in contrast to the prognostic value of MRP1 expression in AML , which has shown conflicting results , whereas currently , LRP is no longer thought to be important for clinical drug resistance -LSB- 4 , 5 , 7 , 24 -- 27 -RSB- . Recently , two studies in , respectively , 40 and 31 adult AML patients showed no effect of BCRP gene expression on CR rate , whereas OS was lower in patients with the highest BCRP expression -LSB- 10 , 23 -RSB- . Damiani et al. -LSB- 28 -RSB- showed that BCRP expression did not influence achievement of complete remission in AML patients with a median age of 53 years and normal karyotype , however , BCRP expression was associated with higher relapse rate . In 59 children with de novo AML , a higher BCRP expression was observed in patients who did not reach CR , but this was not translated in poorer survival -LSB- 29 -RSB- . Benderra et al. -LSB- 19 -RSB- indicated that BCRP gene expression was an adverse prognostic factor for CR in a group of 149 relatively younger adult AML patients but only in patients treated with DNR and MXT and not with idarubicin . In our cohort of elderly AML patients who were all treated with DNR , whereas MXT was given as consolidation therapy after reaching CR , a significant correlation of BCRP mRNA expression with lower CR rate could not be shown . Our study confirms that BCRP and MDR1 are coexpressed in AML patients with higher age as has been suggested previously from studies in smaller groups of relatively younger AML patients -LSB- 9 -- 11 , 28 -RSB- . Until now , only two studies have evaluated the clinical value of coexpression of MDR1 and BCRP in a sufficient number , although relatively younger adult AML patients -LSB- 19 , 28 -RSB- . Benderra et al. -LSB- 19 -RSB- showed that CR rate was only 45 % in the patients with coexpression of BCRP and MDR1 -LRB- + / + -RRB- in comparison with 66 % in the MDR1/BCRP − / + and + / − group and 90 % in the MDR1/BCRP − / − group -LRB- p = 0.003 -RRB- . Moreover , a significantly lower DFS and OS were found in the MDR1/BCRP + / + group . Damiani et al. -LSB- 28 -RSB- found a trend towards a higher relapse rate in the small group of BCRP + / MDR1 + patients , indicating that this represents a robust resistant AML phenotype , consistent with our findings in elderly AML . The recent finding that BCRP and MDR1 expression was mainly found in the most resistant group of AML , using gene expression profiling , underscores the role of these drug resistance genes in AML -LSB- 30 -RSB- . However , this study shows , that the prominent prognostic role of CD34 expression in elderly AML should be emphasized , as higher CD34 expression was adversely associated with all clinical endpoints . MDR1 and BCRP but not MRP1 and LRP mRNA expression were found to be associated with high CD34 expression in these elderly AML patients , which may explain why MDR1 was no longer significant for CR rate , EFS , and OS when adjusted for other prognostic variables including CD34 . In the past , MDR1 expression has been linked to the CD34-positive hematopoietic stem cell compartment of the leukemia subtype . In two other studies in younger AML patients , no overexpression -LRB- on mRNA and protein level -RRB- of BCRP in the CD34-positive blast population of clinical AML samples was found -LSB- 13 , 19 -RSB- . In contrast , earlier studies in mice demonstrated high levels of BCRP and MDR1 expression in normal hematopoietic stem cells -LSB- 31 -- 34 -RSB- . Previously , BCRP expression in subsets of stem cells has been reported , indicating that high BCRP expression may exist in CD34 + / CD38 − cells or in CD34 + / CD33 − cells -LSB- 12 , 35 -RSB- . The differential expression of BCRP and MDR1 in specific subsets of hematopoietic stem cells is consistent with the side population phenotype as proposed by Goodell et al. -LSB- 36 -RSB- who claim that BCRP expression can be separated from those expressing the other ABC proteins . This would suggest that BCRP is expressed in even less differentiated hematopoietic stem cells than MDR1 -LSB- 19 -RSB- . In our study in AML , these immature subsets could not be separately investigated , however , the unique BCRP/MDR1 + / + subgroup of patients reflects an immature leukemic cell type that has a very resistant phenotype in vivo , illustrated by a low CR rate and poor outcome -LRB- Table 3 ; Fig. 2 -RRB- . This is the first study in which a correlation was found between secondary AML and a high expression of BCRP mRNA but not the other resistance proteins . In addition to our previous report that BCRP is frequently upregulated in patients with AML at relapse , we now demonstrate that expression of BCRP is representative of secondary AML , which is especially observed in elderly patients -LSB- 11 , 29 -RSB- . Recently , Ross -LSB- 37 -RSB- suggested that MDR modifiers may be of benefit for patients with multiple dysplastic features . This may suggest that BCRP is upregulated in diseases in which exposure to xenobiotics during life plays an etiologic role . We conclude that coexpression of CD34-related coexpression of MDR1 and BCRP reflects a clinically resistant subgroup of elderly AML . In this age group , only BCRP is correlated with secondary AML . As such , the development of new treatment strategies for elderly AML patients may focus on modulation of drug resistance targeting both BCRP and MDR1 .

O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O B O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O B O B O O B O B O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O B O B O O O O O O O O O O O O O O O O O B O B O B O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O B O B O O O O B O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O B O B O O B O O O O O O O O O O O O O O O O B I O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O B I O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O B O O B O B O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O B O


Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria -LRB- SRB -RRB- in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater . The wastewater was fed with ethanol as an external electron donor . Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth -LRB- i.e. 108 -RRB- . Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity , with only two genotypes among the 26 strains obtained in the pure culture . The low genetic diversity suggests the absence of micro-niches within the reactor , which might be due to a low spatial and temporal micro-heterogeneity . The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio . The two strains differed in as many as five physiological traits , which might allow them to occupy distinct niches and thus co-exist within the same habitat . Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor . The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB , representing 30 -- 35 % and 25 -- 35 % , respectively , of the total SRB community . Desulfobulbus-like bacteria contributed for 20 -- 25 % , and the Desulfobacca acetoxidans-specific probe targeted approximately 15 -- 20 % of the total SRB . The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor . Introduction Many industrial processes , such as metal smelting , flue gas scrubbing and mining generate sulfate-rich wastewater -LRB- Lens et al. 1998 -RRB- . These wastewaters usually do not contain any organic carbon , and the addition of external electron donor is required for their treatment . These waste streams cause a range of problems associated with the now well-established anaerobic treatment of these wastewaters , such as a decrease in methane production -LRB- Colleran et al. 1995 -RRB- , sulfide toxicity -LRB- O'Flaherty and Colleran 2000 -RRB- , foul smell -LRB- Lens et al. 2001 -RRB- and corrosion -LRB- Vincke et al. 2001 -RRB- . Of late , bioreactor processes have been developed for treating these sulfate and metal-rich wastewaters . This technology developed by the company Paques BV in The Netherlands -LRB- Pol et al. 2001 -RRB- makes use of the dissimilatory sulfate-reducing capacity of sulfate-reducing bacteria -LRB- SRB -RRB- to simultaneously remove sulfate and metals in the form of metal sulfides . A typical application is a two-step process that includes dissimilatory sulfate reduction to sulfide -LRB- Gibson 1990 -RRB- as the first step . Subsequently , the sulfide will be bound by heavy metals , such as cobalt , lead , nickel and zinc and precipitated as metal sulfide -LRB- Colleran et al. 1995 -RRB- . The second step is the biological oxidation of the remaining sulfide to insoluble elemental sulfur , which is either recovered by sedimentation or a small portion converted to H2SO4 and re-used to neutralise the alkalinity generated because of sulfide production in the first step . Several full-scale sulfate removal bioreactors are currently in operation . The dominant sulfidogenic communities in these bioreactors are heterotrophic SRB belonging to the family Desulfovibrionaceae -LRB- Kaksonen et al. 2004 ; van Houten et al. 2006 -RRB- . The genus Desulfovibrio represents a phylogenetically coherent group ; all species incompletely oxidise lactate to acetate but can utilise hydrogen , formate and ethanol as well -LRB- Widdel and Bak 1992 -RRB- . In an engineered system fed with a single nutritional source and kept under constant operational parameters such as pH , temperature and salinity , competition among species might tend to reduce the species and sub-species diversity according to the principle of competitive exclusion -LRB- Gause 1934 -RRB- . However , physiologically competing species can co-exist if they occupy different niches , whereby each species uses distinct parts of the resource base . In the present study , we investigated the co-existence of physiologically similar hydrogenotrophic SRB in a full-scale sulfidogenic bioreactor treating sulfate-rich wastewater using a combination of cultivation and molecular techniques . The reactor was fed with ethanol as carbon and energy source . SRB isolated in pure culture were characterised genetically and physiologically . Micro-diversity among the dominant culturable isolates was assessed by repetitive enterobacterial palindromic polymerase chain reaction -LRB- rep-PCR -RRB- -LRB- Versalovic et al. 1994 -RRB- . Whole-cell hybridisation with fluorescently labelled general and specific probes was used for SRB community characterisation and for the estimation of the relative abundance of the different SRB populations in the reactor . Materials and methods Sludge source Granular sludge was obtained from an upflow anaerobic sludge bed -LRB- UASB -RRB- reactor , treating sulfate-rich wastewater from a chemical plant located in Emmen , The Netherlands . The wastewater fed to the reactor did not contain any organic compound , so ethanol was added as an external electron donor and carbon source . The reactor had a volume of 300 m3 and a feed rate of 60 -- 75 m3 h − 1 The ratio between the amount of electron donor added and the amount of sulfate in the reactor was around 0.4 kg/kg . Sulfide produced in the reactor was either converted to elemental sulfur through biological sulfide oxidation or precipitated with toxic metals . The reactor was operated at a temperature of 30 °C and a pH of 7.0 -- 7.5 . The concentration of sulfate in the wastewater was approximately 1,500 mg/l and ca. 100 mg/l in the effluent . Culture media and isolation of SRB A basal bicarbonate-buffered and sulfide-reduced medium was used for the enumeration and isolation procedures . The mineral medium contained per litre of distilled water : 0.2 g KH2PO4 , 0.25 g NH4Cl , 0.5 g KCl , 0.1 g CaCl2 · 2H2O , 0.4 g MgCl2 · 6H2O , 1.0 g NaCl and 0.5 ml of a resazurin solution -LRB- 0.5 mg ml − 1 -RRB- as a redox indicator . The medium was supplemented with -LRB- per litre -RRB- the following : 30 ml 1 M NaHCO3 solution , 1 ml of a vitamin solution , 1 ml of trace element solution with ethylenediamine tetraacetic acid -LRB- EDTA ; Widdel and Bak 1992 -RRB- and 0.1 g of yeast extract . As a reducing agent , 7.5 ml l − 1 of 0.2 M Na2S · 9H20 was added . Either lactate or ethanol -LRB- 20 mM -RRB- was used as an electron donor and sulfate -LRB- 10 mM -RRB- as electron acceptor . Enumeration of the potentially dominant heterotrophic sulfate reducers was performed by serial dilutions in Hungate tubes . The Hungate tubes were incubated at 30 °C in the dark for 7 weeks . The highest positive dilution tubes that showed growth were selected for further isolation . Growth was assayed by measuring sulfide production photometrically -LRB- Cord-Ruwisch 1985 -RRB- . Pure cultures were obtained by repeated transfer in agar shake tubes -LRB- Widdel and Pfennig 1984 -RRB- . Purity of the isolates was checked by microscopic observation and further confirmed by denaturing gradient gel electrophoresis -LRB- DGGE -RRB- analysis of PCR-amplified 16S rRNA gene fragments -LRB- Teske et al. 1996 -RRB- . Phenotypic characterisation Substrate utilisation by the isolated strains was determined in duplicate in the same bicarbonate-buffered medium as used for enumeration and isolation but without yeast extract . Growth was tested in 100-ml serum bottles closed with butyl rubber stoppers and aluminium crimp seals . The inoculum size was 1 % -LRB- v/v -RRB- . The cultures were incubated at 30 °C for 5 weeks in the dark . Growth was determined by substrate consumption or product formation as well as by checking for increase in optical density at 660 nm -LRB- OD660 -RRB- . The following substrates were tested as electron donors in the presence of 10 mM sulfate : 10 mM each of pyruvate , fumarate , butyrate , formate , propanol , ethanol , methanol , serine and cysteine ; 5 mM each of malate , glycolate and glycerol ; 2.5 mM of benzoate ; and 0.5 % -LRB- w/v -RRB- of casamino acids . Sulfite , thiosulfate and 2 % -LRB- w/v -RRB- elemental sulfur were tested as electron acceptors in the presence of lactate as electron donors . For measuring catalase activity , the fully grown isolates were centrifuged at 13,000 × g . The cell pellets were re-suspended on glass slides with a drop of 3 % -LRB- v/v -RRB- H2O2 , bubbles indicated the presence of catalase . Detection of desulfoviridin was performed according to Postgate -LRB- 1959 -RRB- . Gram staining was performed as previously described -LRB- Bartholomew 1962 -RRB- . Analytical methods Acetate and other volatile fatty acids were analysed either by gas chromatography -LRB- GC -RRB- or high performance liquid chromatography -LRB- HPLC -RRB- . For GC , a Chromopack 9001 , equipped with a flame ionisation detector and a fused-silica capillary column 15 × 0.53 mm HP-Innowax , was used . The column temperature was 120 °C . The temperature of the injector and detector were 180 and 200 °C , respectively . Helium was used as a carrier gas . An Aminex HPX-87H column from Bio-Rad -LRB- T = 60 °C -RRB- coupled to a UV and a RI detector was used for HPLC ; phosphoric acid -LRB- 0.05 M -RRB- was used as an eluent . Sulfide was measured quantitatively by a colorimetric assay -LRB- Cline 1969 -RRB- . DNA extraction Genomic DNA was isolated from the bacterial cultures using the Ultra Clean Soil DNA extraction kit -LRB- MOBIO Laboratories , California -RRB- according to the manufacturer 's protocol . The quality of the extracted DNA was examined on a 1 % -LRB- w/v -RRB- agarose gel and the amount quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM -LRB- NanoDrop Technologies , Delaware -RRB- . Extracted DNA was stored at − 20 °C until subsequent use in different PCR reactions . PCR amplification and DGGE of 16S rRNA genes For DGGE analysis , amplification of partial 16S rRNA gene was carried out using the primers 341F-GC and 907R as described by -LRB- Schäfer and Muyzer 2001 -RRB- , while primers GM3 and GM4 -LRB- Muyzer et al. 1995 -RRB- were used to amplify the nearly complete 16S rRNA gene for sequencing and subsequent phylogenetic analysis . PCR amplification and DGGE was performed as described previously -LRB- Schäfer and Muyzer 2001 -RRB- . The quality of the PCR products was examined on 1 % -LRB- w/v -RRB- agarose gel , and the yield was quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM -LRB- NanoDrop Technologies -RRB- . DNA sequencing and phylogenetic analysis The nearly complete 16S rRNA gene fragments , obtained from the strains L3 and L7 , were purified using the Qiaquick Gel Extraction Kit -LRB- Qiagen , Hilden , Germany -RRB- . Purified PCR products were sequenced by the company BaseClear -LRB- Leiden , The Netherlands -RRB- . The DNA sequences of about 1,400 bp were first compared to the sequences deposited in public databases using the NCBI BLAST search tool -LRB- http://www.ncbi.nlm.nih.gov/BLAST ; McGinnis and Madden 2004 -RRB- . Subsequently , the sequences were imported into the ARB software programme -LRB- Ludwig et al. 2004 -RRB- and aligned using the automatic aligner function . The alignment was further corrected manually , and a phylogenetic tree was constructed using the neighbour-joining algorithm with Felsenstein correction . rep-PCR fingerprinting The genetic diversity of the isolates was analysed by rep-PCR -LRB- Versalovic et al. 1994 -RRB- using the primer GTG5 -LRB- 5O ′ - gTggTggTggTggTg-3 ′ -RRB- . The amplification reaction was performed as previously described -LRB- Foti et al. 2006 -RRB- . A 1-kb size marker and 600 ng of the PCR product were loaded onto a 1.5 % -LRB- w/v -RRB- agarose gel containing 0.5 × TAE-buffer -LRB- 200 mM Tris-acetate , 0.5 mM EDTA , pH 8 -RRB- . The electrophoresis was performed for 14 h in a cold room at a constant voltage of 65 V . The gel was subsequently stained with ethidium bromide -LRB- 0.5 μg/ml -RRB- and photographed under UV illumination using the GelDoc UV Transilluminator -LRB- Bio-Rad , Hercules , CA -RRB- . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein -LRB- in cell-free extract obtained by sonication -RRB- was performed on 12 % polyacrylamide gels according to Laemmli -LRB- 1970 -RRB- . The gels were stained with Coomassie Brilliant Blue and photographed using visible light . Design of oligonucleotide probes Specific oligonucleotide probes for the 16S rRNA of the two strains were designed using the Probe Design tool of the ARB software package -LRB- Ludwig et al. 2004 -RRB- . The requirements for designing the probes included high specificity , with no organism outside the intended target group having 100 % similarity within the target sequence , and the target sequence being located within the high accessibility region of 16S rRNA molecule as suggested previously -LRB- Behrens et al. 2003 -RRB- . The probes were named with a number that indicates the position of the first base in the target sequence -LRB- by Escherichia coli numbering -RRB- . The oligonucleotides used for in situ hybridisation are given in Table 1 . Table 116S rRNA-targeted oligonucleotide probes used in this studyProbe nameaTarget organismsProbe sequence -LRB- 5 ′ -- 3 ′ -RRB- ReferenceEUB338_IMost bacteriaGCT GCC TCC CGT AGG AGTAmann et al. -LRB- 1990 -RRB- EUB338_IIPhylum PlanctomycetesGCA GCC ACC CGT AGG TGTDaims et al. -LRB- 1999 -RRB- EUB338_IIIPhylum VerrucomicrobiaGCT GCC ACC CGT AGG TGTDaims et al. -LRB- 1999 -RRB- ARCH915ArchaeaGTG CTC CCC CGC CAA TTCStahl and Amann -LRB- 1991 -RRB- SRB385Most DeltaproteobacteriaCGG CGT CGC TGC GTC AGGAmann et al. -LRB- 1990 -RRB- SRB385DbSome DeltaproteobacteriaCGG CGT TGC TGC GTC AGGRabus et al. -LRB- 1996 -RRB- DSR660Genus DesulfobulbusGAA TTC CAC TTT CCC CTC TGDevereux et al. -LRB- 1992 -RRB- DSBA1017Desulfobacca acetoxidansGTT GCC AGG CAC CCC CATDar et al. -LRB- 2007 -RRB- DSV119Desulfovibrio strain L3GGC AGA TCA TCC ACG CGTThis studyDSV139Desulfovibrio strain L7CGC TGT TAT CCC GAT CACThis studyaEUB338 is a combination of EUB338_I , EUB338_II and EUB338_III . Whole cell hybridisation Cells from strains L3 and L7 and from the original reactor were fixed , washed and spotted onto Teflon-coated multi-well microscopic slides as described previously -LRB- Dar et al. 2007 -RRB- . Hybridisation was carried out according to the protocol described by Manz et al. -LRB- 1992 -RRB- using a formamide concentration of 35 % -LRB- v/v -RRB- . Quantification of the hybridised cells was performed as described previously -LRB- Neef et al. 1996 -RRB- . The hybridised cells were analysed by two independent observers for determining the fraction of positive signal from each probe relative to the signal visualised with general probes for bacteria -LRB- EUB338 I , II and III -RRB- , SRB -LRB- SRB385 and SRB385Db -RRB- or with the general DNA stain DAPI -LRB- 4 ′ , 6 ′ - diamidino-2-phenylindole -RRB- . In addition , a general probe specific for members of the domain Archaea -LRB- ARCH915 -RRB- was used . The hybridisation experiments were done in duplicate using different fluorochromes for each probe . Different microscopic fields on each slide were analysed to confirm the results . Hybridisation stringencies of the newly designed probes were determined by performing hybridisations with increasing formamide concentrations as described previously -LRB- Manz et al. 1992 -RRB- using target organism -LRB- s -RRB- and non-target organism displaying three mismatches within the target region . Sequence accession numbers The nearly complete rRNA gene sequences of strains L3 and L7 have been deposited in GenBank under accession nos . EF055876 and EF055877 , respectively . Results Reactor performance The amount of ethanol dosed to the reactor was continuous and limited to the amount required for sulfate reduction according to the net stoichiometric reaction for sulfidogenic oxidation of ethanol and its major degradation intermediate , acetate . The equations are as follows : The sulfate removal efficiency at the time of sampling was more than 93 % . Less than 1 mg of acetate was estimated in the effluent and no significant biogas -LRB- CH4 -RRB- production from the oxidation of organic source was observed at the time of sampling . Enumeration and isolation of SRB The maximum number of culturable SRB observed with lactate or ethanol as substrate in the serial dilution tubes was in the order of 108 cells ml − 1 . The tubes with growth in the highest dilution were used for isolation in agar shake tubes . In total , 26 strains were obtained in pure culture , 17 on lactate and nine on ethanol as substrate . Sub-cultivation of the isolated strains was carried out on lactate . Apart from microscopic observation , the purity of the isolated strains was confirmed by single bands obtained from the DGGE of partial 16S rRNA genes amplified from the strains . The DGGE results of the 26 isolated strains identified two melting types -LRB- results not shown -RRB- . Genomic fingerprinting The isolated strains were subjected to genomic fingerprinting , i.e. rep-PCR , to resolve any higher degree of genetic diversity among the strains . The rep-PCR profiles of the strains -LRB- Fig. 1 -RRB- revealed the presence of two distinct genotypes with no micro-diversity among the strains . Furthermore , the whole cell protein profile of representative strains of the two groups , grown on the same substrate -LRB- i.e. lactate and sulfate -RRB- and under similar conditions of pH and temperature , gave only two distinct profiles -LRB- Fig. 2 -RRB- , confirming the presence of two genotypes . Two isolates , strain L3 and L7 , were chosen as representatives of the two genotypes and were subjected to a more detailed phylogenetic and phenotypic analysis . Fig. 1rep-PCR patterns of different strains isolated from the full-scale sulfidogenic bioreactor . M is the molecular weight markerFig . 2Whole cell protein profile of different strains isolated from the full-scale sulfidogenic bioreactor . M is the molecular weight marker . The arrows depict protein bands that are expressed in one or the other genotype Phylogenetic analysis A similarity check using the NCBI BLAST search tool of the nearly complete 16S rRNA gene sequences obtained from strain L3 and L7 indicated 98 % sequence similarity to Desulfovibrio strain SB1 and 99 % sequence similarity to Desulfovibrio mexicoense , respectively . The phylogenetic affiliation of the obtained sequences is presented in Fig. 3 . A neighbour-joining tree based on nearly complete 16S rRNA gene sequences was generated , confirming the close affiliation of the isolated strains L3 and L7 to Desulfovibrio strain SB1 and Desulfovibrio mexicoense , respectively . Fig. 3Neighbour-joining tree based on nearly complete 16S rRNA gene sequences showing the phylogenetic affiliation of the two isolated SRB , strain L3 and L7 . The sequence of Desulfobacter postgatei was used as an outgroup but was pruned from the tree . Dots on the nodes indicate bootstrap values of 90 % or higher -LRB- 1,000 replicates -RRB- . The bar indicates 1 % sequence difference Phenotypic characterisation of the strains All isolates had a vibrio to spiral cell morphology . Two distinct groups could be identified based on size and motility . One group of cells related to Desulfovibrio strain SB1 was motile , and their size ranged from 4 -- 7 μm in length and 1 μm in width . The other group was non-motile , and the size was 1 -- 2 μm in length and 0.5 μm in width . Table 2 shows the substrate utilisation patterns of the two isolates . Both strains used lactate , ethanol , pyruvate , glycerol and casamino acids as electron donors . Hydrogen and formate could only be used as substrates in the presence of acetate as carbon source . The organic substrates were incompletely oxidised to acetate . Acetate , propionate , butyrate , glycolate and methanol could not be used as electron donors by both strains . Fumarate and malate were used by strain L3 but not by strain L7 , while serine and cysteine were utilised by strain L7 but not by strain L3 . Sulfate , thiosulfate and sulfite were used as electron acceptors by both strains . In addition , strain L7 could also use elemental sulfur as an electron acceptor . Table 2Phenotypic characterisation of strains L3 and L7CharacteristicsStrain L3Strain L7Cell morphologyVibrioVibrioMotility + − Desulfoviridin + + Catalase + + Gram staining − − Electron donors H2 plus acetate + + Pyruvate + + Lactate + + Acetate − − Propionate − − Fumarate + − Butyrate − − Formate + + Propanol − − Ethanol + + Methanol − − Serine − + Cysteine − + Malate + − Glycolate − − Glycerol + + Benzoate − − Casamino acids + + Electron acceptors Thiosulfate + + Sulfite + + Elemental sulfur − + Whole cell hybridisation After isolation of the most abundant culturable SRB , whole cell hybridisation with specific oligonucleotide probes was performed to estimate the abundance of strains L3 and L7 in the original sludge sample . The specificity of the designed oligonucleotide probes -LRB- DSV119 and DSV139 -RRB- was verified using growing cells of strain L3 and Desulfovibrio gigas for probe DSV119 and cells of strain L7 and Desulfovibrio mexicoense for probe DSV139 . Strain L3 served as a non-target species for L7 and vice versa . A formamide concentration of 35 % -LRB- v/v -RRB- was found stringent enough to discriminate between the two strains -LRB- Fig. 4a -RRB- . In addition , probes specific for cells of Desulfobulbus -LRB- i.e. DSR660 -RRB- and Desulfobacca acetoxidans -LRB- i.e. DSBA1017 -RRB- , which we had detected previously in a similar sulfidogenic reactor -LRB- Dar et al. 2007 -RRB- , were also used . Fig. 4a Mixture of cells of strain L3 and L7 hybridised with probe Dsv139 labelled with Cy5 -LRB- blue -RRB- , probe Dsv119 labelled with Cy3 -LRB- red -RRB- and probe SRB385 labelled with Fluos -LRB- green -RRB- . b Sludge sample from a full-scale sulfidogenic bioreactor hybridised with probe Dsv139 labelled with Cy3 -LRB- red -RRB- , probe Dsv119 labelled with Cy5 -LRB- blue -RRB- and SRB385 labelled with Fluos -LRB- green -RRB- . c Sludge sample hybridised with probe Dsv139 labelled with Cy5 -LRB- blue -RRB- , probe Dsv119 labelled with Cy3 -LRB- red -RRB- and EUB338 labelled with Fluos -LRB- green -RRB- . d Sludge sample hybridised with probe DSR660 labelled with Fluos -LRB- green -RRB- , probe SRB385 labelled with Cy3 -LRB- red -RRB- and EUB338 labelled with Cy5 -LRB- blue -RRB- . e Sludge sample hybridised with probe SRB385 labelled with Fluos -LRB- green -RRB- , probe DSBA1017 labelled with Cy3 -LRB- red -RRB- and probe EUB338 labelled with Cy5 -LRB- blue -RRB- . f Sludge sample hybridised with probe Arch915 labelled with Fluos -LRB- green -RRB- , probe SRB385 labelled with Cy3 -LRB- red -RRB- and EUB338 labelled with Cy5 -LRB- blue -RRB- . Bar is 20μm The relative percentage of cells that hybridised with probe DSV119 , which is specific for strain L3 , was 30 -- 35 % of the total SRB385-positive cells and 15 -- 20 % of the EUB338-positive cells -LRB- Table 3 ; Fig. 4b , c -RRB- . The probe specific for strain L7 , i.e. DSV139 , was detected between 25 -- 35 % of the SRB385-positive cell and 10 -- 20 % of the EUB338-positive cells -LRB- Table 3 ; Fig. 4b , c -RRB- . The Desulfobulbus and Desulfobacca-specific probes also gave a positive signal , but these SRB were somewhat less abundant than Desulfovibrio positive cells . The Desulfobulbus-specific probe , DSR660 , targeted approximately 20 -- 25 % of the total SRB cells and 10 -- 15 % of the total bacterial cells -LRB- Table 3 ; Fig. 4d -RRB- . The Desulfobacca acetoxidans-specific probe DSBA1017 targeted approximately 15 -- 20 % of the total SRB cells and 8 -- 10 % of bacterial cells -LRB- Table 3 ; Fig. 4e -RRB- . The percentage of cells that hybridised with probe specific for Archaea -LRB- probe ARCH915 -RRB- was less than 0.1 % of the total DAPI stained cells -LRB- Table 3 ; Fig. 4f -RRB- . Table 3Relative abundance of SRB and ArchaeaaProbeSRB385EUB338DAPIDSV11930 -- 3515 -- 2012 -- 16DSV13925 -- 3510 -- 208 -- 16DSR66020 -- 2510 -- 158 -- 12DSBA101715 -- 208 -- 106 -- 8ARCH915 -- -- < 0.1 aPercentage of positive cells relative to those detected by a mixture of probes SRB385 and SRB385Db -LRB- SRB385 -RRB- , a mixture of probes EUB338 I , II and III -LRB- EUB338 -RRB- and to cells stained with the DNA stain DAPI . Discussion Based on the observed sulfate removal efficiency -LRB- > 93 % -RRB- and the amount of ethanol dosed to the reactor , it may be assumed that a major part of electron flow from the substrate is scavenged by the SRB . The presence of less than a milligram of acetate in the effluent stream with no significant production of CH4 points to the oxidation of ethanol mainly through sulfate reduction , although fermentation of ethanol through acetogenesis can not be ruled out . The metabolic products of fermentation and acetogenesis , mainly acetate and hydrogen , may serve as substrates for methanogens and sulfate reducers ; however , under high sulfate concentrations in the reactor , hydrogen and acetate would be more readily used by hydrogenotrophic and acetate-utilising sulfate reducers , respectively , because of more favourable substrate affinity -LRB- Ks -RRB- values of the SRB for these substrates -LRB- Stams et al. 2005 -RRB- . Fermentation of ethanol with propionate as the major reduced end product has also been described in the literature -LRB- Schink 1984 ; Tholozan et al. 1992 -RRB- ; and under such fermentation , propionate will serve as an energy source for the members of the genus Desulfobulbus among the SRB . Genetic diversity among the dominant culturable isolates Compared to high micro-diversity among phylogenetically similar strains detected in sediments -LRB- Sass et al. 1998 ; Wieringa et al. 2000 ; Klepac-Ceraj et al. 2004 -RRB- , no such micro-diversity was observed within the 26 strains isolated from the bioreactor . The chemical complexity of sediments with steep gradients of substrate concentrations , redox potential and pH may give rise to a number of physico-chemical and depth-defined micro-niches , resulting in the evolution of co-existing but genetically distinct sub-populations -LRB- Gray et al. 1999 -RRB- . In addition , Torsvik et al. -LRB- 2002 -RRB- suggested structural complexity of sediments being an important factor that allows nutritional partitioning , creating numerous niches that , in turn , results in genetic diversification of populations . From the 26 strains isolated , the ones that had identical 16S rRNA gene sequences were also identical by genomic fingerprinting -LRB- Fig. 1 -RRB- and whole cell protein electrophoresis -LRB- Fig. 2 -RRB- . Based on the rep - and protein profiles , two genotypes could be identified among the 26 strains isolated . The absence of micro-diversity among the dominant culturable Desulfovibrio populations suggests the existence of a few ecological niches in the reactor . The long-term operation of the reactor under stable operational parameters , like constant temperature , pH , salinity etc. , combined with the selection pressure because of nutritional limitation -LRB- i.e. the use of a single energy source -RRB- , might be the reason for absence of micro-diversity . In addition , the upward flow of wastewater through the reactor results in frequent mixing , which , in turn , might prevent the creation of micro-habitats that are assumed to be important for the evolution of genetically distinct sub-populations -LRB- Torsvik et al. 2002 -RRB- . Although the viable cell count of SRB observed -LRB- 108 cell ml − 1 -RRB- was comparable to the previous studies -LRB- Vester and Ingvorsen 1998 ; Oude Elferink et al. 1999 ; Roest et al. 2005 ; van Houten 2006 -RRB- , it can not be ruled out that potential biases associated with culture-based enumeration techniques might have underestimated the overall SRB population diversity . Phenotypic and phylogenetic characterisation of the dominant culturable isolates The physiological characteristics -LRB- Table 2 -RRB- of strain L3 and L7 indicated that they are sulfate reducers . The two isolates oxidised lactate and ethanol incompletely to acetate in the presence of sulfate as electron acceptor . Both used H2 and formate in the presence of acetate as carbon source . These metabolic traits , in addition to the presence of desulfoviridin and a typical vibrio-shaped morphology , suggested that they are the members of the genus Desulfovibrio -LRB- Widdel and Bak 1992 -RRB- . Desulfovibrio are Gram-negative sulfate reducers , most of which oxidise their substrates incompletely to acetate -LRB- Widdel and Bak 1992 -RRB- . Previous studies have demonstrated the dominance of Desulfovibrio species in freshwater sediments -LRB- Sass et al. 1998 -RRB- , in oil wells -LRB- Voordouw et al. 1996 -RRB- and in several wastewater treatment plants -LRB- Santegoeds et al. 1998 ; Dar et al. 2005 -RRB- . 16S rRNA sequence analysis of the two strains , L3 and L7 , confirmed their affiliation to other members of the genus Desulfovibrio -LRB- Fig. 3 -RRB- , with Desulfovibrio strain SB1 and Desulfovibrio mexicoense as closest relatives , respectively . Desulfovibrio strain SB1 , a mesophilic , Gram-negative SRB was isolated from anaerobic sludge of a gas lift reactor-treating sulfate and zinc-rich wastewater -LRB- van Houten 2006 -RRB- ; while Desulfovibrio mexicoense was isolated from a UASB digester-treating wastewater from a cheese-manufacturing factory in Mexico -LRB- Hernandez-Eugenio et al. 2000 -RRB- . Co-existing SRB populations in the reactor After isolation of the most abundant culturable SRB , i.e. the motile strain L3 and non-motile strain L7 , whole cell hybridisation using fluorescently labelled oligonucleotide probes was performed to search for their specific signals in the fixed sludge sample . Hybridisation results not only confirmed the presence of the two isolates but also gave an estimate of their abundance relative to the total SRB population and to the overall bacterial community present -LRB- Table 3 ; Fig. 4 -RRB- . Fluorescence in situ hybridisation -LRB- FISH -RRB- results indicated that the two isolates indeed made up a major part of the hydrogenotrophic SRB community present in the reactor . Cells detected by probe Dsv119 , specific for the motile strain L3 , appeared as single cells or as chains of three to four cells , while cells detected by probe Dsv139 , specific for the non-motile strain L7 , appeared as individual cells or as loose aggregates . According to the ecological principle of competitive exclusion , co-existence of physiologically related populations in the same habitat can be understood if they occupy distinct ecological niches -LRB- Gause 1934 -RRB- . Although a number of similarities can be drawn between the strains , L3 and L7 , from their substrate utilisation profiles -LRB- Table 2 -RRB- , the phenotypic differences together with differences in the range of substrates used by the two isolates may allow them to adapt to slightly different niches within the reactor . The two isolates differed in as many as five physiological properties , which are the use of malate , formate , serine and cysteine as energy sources and sulfur as an electron acceptor -LRB- Table 2 -RRB- . The use of elemental sulfur in particular by strain L7 will give it a selective advantage over strain L3 in those places in the reactor where elemental sulfur might be available because of the re-circulation fluid from the aerobic reactor ; whereas the motility of strain L3 will confer a competitive advantage to it over strain L7 . Potential benefits of motility may include increased efficiency of nutrient acquisition and avoidance of toxic substances -LRB- An et al. 2006 -RRB- . The presence of Desulfobulbus-like SRB besides the members of Desulfovibrio in the same habitat could be explained because of the ability of Desulfobulbus to use the fermentation product propionate as an energy source as well ; furthermore , under limiting sulfate concentrations , Desulfobulbus competes more successfully for ethanol than other sulfate reducers by its ability to ferment ethanol -LRB- Laanbroek et al. 1982 -RRB- . The simultaneous presence of Desulfovibrio and Desulfobulbus in wastewater treatment systems has often been reported in the literature -LRB- Nanninga and Gottschal 1987 ; Raskin et al. 1995 ; Okabe et al. 2003 -RRB- . Among the complete oxidisers , the probe specific to Desulfobacca acetoxidans gave a positive signal , suggesting their dominance among the acetotrophic sulfate reducers . Desulfobacca acetoxidans , first isolated from a sulfidogenic bioreactor -LRB- Oude Elferink et al. 1999 -RRB- , is a Gram-negative SRB that can utilise acetate as the only source of organic carbon and electron donor . Kaksonen et al. -LRB- 2004 -RRB- also found Desulfobacca acetoxidans-like SRB in their lab-scale fluidised-bed reactors that were fed with a single electron donor , i.e. lactate or ethanol . In summary , this study demonstrated the presence of a consortium of four sulfate-reducing populations in the reactor maintained on a single substrate -LRB- ethanol -RRB- . This is in contrast to the findings of Kaksonen et al. -LRB- 2004 -RRB- who reported a relatively more diverse consortium of SRB in a fluidised-bed reactor fed with ethanol or lactate as the only energy source , using clone libraries and DGGE as molecular methods for microbial characterisation . The observed difference in the extent of diversity might be due to the difference in the molecular methods employed . The general probes like DSR660 , used during FISH analysis , can not discriminate among the different species , and the probes like DSV119 , DSV139 and DSBA1017 target only the specific populations . A likely complete oxidation of ethanol through sulfate reduction might thus be assumed to be taking place through a combined effort of hydrogenotrophic sulfate reducers -LRB- i.e. the motile strain L3 , the non-motile strain L7 and Desulfobulbus spp. -RRB- oxidising ethanol incompletely to acetate and Desulfobacca acetoxidans oxidising acetate completely to CO2 . Genetic diversity analysis of the most dominant culturable Desulfovibrio populations suggests that long-term stable operation of a reactor with constant operational parameters and frequent mixing might result in the absence of micro-diversity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

sulfate-reducing bacteria
microbial ecology