Detection and quantification of sulfate reducing and sulfur oxidizing bacteria … ·...
1
Chair of Urban Water Systems Engineering, TU München Background TU München, Chair of Urban Water Systems Engineering, Prof. Dr. Brigitte Helmreich, Am Coulombwall, 85748 Garching, Germany, Email: [email protected], Homepage: www.wga.bv.tum.de Material and Methods Funding Objectives BSA in a Digester TECHNISCHE UNIVERSITÄT MÜNCHEN Key Enzymes References Detection and quantification of sulfate reducing and sulfur oxidizing bacteria in wastewater systems using quantitative real-time PCR technique Bettina Huber 1 , Brigitte Helmreich 1 , Rolf König 2 , Elisabeth Müller 1 1 Chair of Urban Water Systems Engineering, Am Coulombwall, 85748 Garching, Germany 2 Weber-Ingenieure GmbH, Stuttgarter Straße 115, 70469, Stuttgart, Germany The objective of this research project is the development of a standardized biochemical test system for the accurate determination of the BSA potential in wastewater systems, especially in digesters. This test system will include: • Detection and identification of SRB and SOB in the digested sludge and aerobic biofilm growing on the concrete surface, respectively • Quantification of the relevant SRB and SOB involved in the BSA process using quantitative real-time PCR-technique (qPCR) Generally, correlations between numbers and types of sulfur-related organisms detected by qPCR and the rate of corrosion shall be investigated. Quantified information shall be gained about the extent of damage so that further evaluations about the structure stability can be made. Concrete corrosion caused by biogenic sulfuric acid is a severe problem affecting the world´s sewerage infrastructure (e.g. concrete sewer pipes) and wastewater treatment (e.g. digesters). Globally, 20% of the total damage of concrete structures in sewer systems seems to be caused by biogenic sulfuric acid corrosion (BSA) leading to repair costs of several billions of dollars per year [1]. For the BSA process, a complex microbial ecosystem, comprising sulfate reducing and sulfur oxidizing bacteria (SRB and SOB, respectively), is responsible. The activity in the wastewater systems creates a sulfur cycle which can lead to bacterial formation of sulfuric acid (H 2 SO 4 ) and consequently to corrosion of concrete (Figure 1 and 2). Figure 1. Schematic of the corrosion process within a sewer [2]. Despite ubiquitous occurrence of BSA, there is no precise method of detection available. Currently, the determination of BSA attacks is carried out optically or by acid analysis. This research project is funded by the Bundesministerium für Wirtschaft und Technologie (BMWi); Zentrales Innovationsprogramm Mittelstand, ZIM-Kooperationsprojekt, AiF Projekt GmbH. [1] Vincke, E., Boon, N., Verstraete, W. (2001). Analysis of the microbial communities on corroded concrete sewer pipes- a case study. Appl Microbiol Biotechnol. 57, 776-785. [2] Wells, T., Melchers, R.E., Bond, P. (2009). Factors involved in the long term corrosion of concrete sewers. In: Australasian Corrosion Association Proceedings of Corrosion and Prevention 2009. Corrosion & Prevention - 2009, Coffs Harbour, Australia. Link: http://hdl.handle.net/1959.13/92006. [3] Ben-Dov, E., Brenner, A., Kushmaro, A. (2007). Quantification of sulfate-reducing bacteria in industrial wastewater, by real-time polymerase chain reaction (PCR) using dsrA and apsA genes. Microbial Ecology. 54, 439-451. [4] Grein, F., Raquel Ramos, A., Venceslau, S.S., Pereira, I. A.C. (2013). Unifying concepts in anaerobic respiration: Insights from dissimilatory sulfur metabolism. BBA- Bioenergetics. 145-160. [5] Friedrich, C.G. Bardischewsky, F., Rother, D., Quentmeier, A., Fischer, J. (2005). Prokaryotic sulfur oxidation. Curr Opin Microbiol. 8, 253-259. [6] Tourova, T.P., Slobodova, N.V., Bumazhkin, B.K., Kolganova, T.V., Muyzer, G., Sorokin, D.Y. (2012). Analysis of community composition of sulfur-oxidizing bacteria in hypersaline and soda lakes using soxB as a functional molecular marker. Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. [7] Petri, R., Podgorsek, L., Imhoff, J.F. (2001). Phylogeny and distribution of the soxB gene among thiosulfate-oxidizing bacteria. FEMS Microbiology Letters. 197, 171-178. 1) Sample collection from a digester: • Anaerobic digested sludge samples containing SRB • Aerobic biofilm removed from concrete surface containing SOB 2) Isolation of genomic DNA from environmental samples: direct cell lysis with glass beads and DNA extraction using phenol chloroform method 3) Detection and identification of relevant SRB and SOB by PCR, PCR- DGGE, phylogenetic sequence analysis and FISH 4) Quantification of SRB and SOB using qPCR technique: selection of specific primer systems from literature data or as needed new primer design and evaluation of qPCR conditions. SRB (heterotrophic): e.g. Desulfovibrio, Desulfobacter SOB (autotrophic): e.g. Thiobacillus, Paracoccus Anaerobic biofilm Aerobic biofilm Condensate film Abiotic corrosion Biotic corrosion CO 2 + H 2 O → H 2 CO 3 H 2 S → H + + HS - H 2 S + 2O 2 SOB H 2 SO 4 Wastewater Liquid-gas exchange Sewer headspace Figure 2. Severe BSA attack in a digester. © R. König 2 e - AMP Sat APS reductase (ApsAB) Dissimilatory sulfite reductase DsrAB/ DsrC Sulfate Adenosine 5´-phosphosulfate (APS) Sulfite Sulfide ATP 2Pi PPi 6 e - Figure 3. Dissimilatory sulfate reduction [4]. • Dsr and aps genes (Figure 3) are present in all known sulfate reducing prokaryotes and have been proposed to be useful phylogenetic markers for SRB. • Specific primers detecting dsrA and apsA genes have been used in a qPCR SybrGreen approach [3] and will be applied in the present study. Functional molecular markers for SRB • SOB can be detected and identified using the multi-enzyme Sox (sulfur-oxidizing) complex consisting of SoxB, SoxXA, SoxYZ and SoxCD components [5]. • Sox pathway is described in photo- and chemotrophic SOB that oxidize sulfide and thiosulfate to sulfate [6]. H 2 S + 2 O 2 → SO 4 2- + 2 H + S 2 O 3 2- + H 2 O + 2 O 2 → 2 SO 4 2- + 2 H + • Primer system using the soxB gene which encodes the sulfate thiohydrolase, a key enzyme of the Sox pathway, has been applied in PCR (DNA fragment > 1000bp, [7]). For detecting soxB genes by qPCR a new approach has to be designed and evaluated. Functional molecular markers for SOB
Transcript of Detection and quantification of sulfate reducing and sulfur oxidizing bacteria … ·...
Chair of Urban Water Systems Engineering, TU München
Background
TU München, Chair of Urban Water Systems Engineering, Prof. Dr. Brigitte Helmreich, Am Coulombwall, 85748 Garching, Germany, Email: [email protected], Homepage: www.wga.bv.tum.de
Material and Methods
Funding
Objectives
BSA in a Digester
TECHNISCHE
UNIVERSITÄT
M Ü N C H E N
Key Enzymes
References
Detection and quantification of sulfate reducing and sulfur oxidizing bacteria in wastewater systems using quantitative real-time PCR technique
Bettina Huber1, Brigitte Helmreich1, Rolf König2, Elisabeth Müller1
1Chair of Urban Water Systems Engineering, Am Coulombwall, 85748 Garching, Germany 2Weber-Ingenieure GmbH, Stuttgarter Straße 115, 70469, Stuttgart, Germany
The objective of this research project is the development of a standardized biochemical test system for the accurate determination of the BSA potential in wastewater systems, especially in digesters.
This test system will include: • Detection and identification of SRB and SOB in the digested sludge and
aerobic biofilm growing on the concrete surface, respectively • Quantification of the relevant SRB and SOB involved in the BSA process
using quantitative real-time PCR-technique (qPCR)
Generally, correlations between numbers and types of sulfur-related organisms detected by qPCR and the rate of corrosion shall be investigated. Quantified information shall be gained about the extent of damage so that further evaluations about the structure stability can be made.
Concrete corrosion caused by biogenic sulfuric acid is a severe problem affecting the world´s sewerage infrastructure (e.g. concrete sewer pipes) and wastewater treatment (e.g. digesters). Globally, 20% of the total damage of concrete structures in sewer systems seems to be caused by biogenic sulfuric acid corrosion (BSA) leading to repair costs of several billions of dollars per year [1]. For the BSA process, a complex microbial ecosystem, comprising sulfate reducing and sulfur oxidizing bacteria (SRB and SOB, respectively), is responsible. The activity in the wastewater systems creates a sulfur cycle which can lead to bacterial formation of sulfuric acid (H2SO4) and consequently to corrosion of concrete (Figure 1 and 2). Figure 1. Schematic of the corrosion process within a sewer [2].
Despite ubiquitous occurrence of BSA, there is no precise method of detection available. Currently, the determination of BSA attacks is carried out optically or by acid analysis.
This research project is funded by the Bundesministerium für Wirtschaft und Technologie (BMWi); Zentrales Innovationsprogramm Mittelstand, ZIM-Kooperationsprojekt, AiF Projekt GmbH.
[1] Vincke, E., Boon, N., Verstraete, W. (2001). Analysis of the microbial communities on corroded concrete sewer pipes- a case study. Appl Microbiol Biotechnol. 57, 776-785.
[2] Wells, T., Melchers, R.E., Bond, P. (2009). Factors involved in the long term corrosion of concrete sewers. In: Australasian Corrosion Association Proceedings of Corrosion and Prevention 2009. Corrosion & Prevention - 2009, Coffs Harbour, Australia. Link: http://hdl.handle.net/1959.13/92006.
[3] Ben-Dov, E., Brenner, A., Kushmaro, A. (2007). Quantification of sulfate-reducing bacteria in industrial wastewater, by real-time polymerase chain reaction (PCR) using dsrA and apsA genes. Microbial Ecology. 54, 439-451.
[4] Grein, F., Raquel Ramos, A., Venceslau, S.S., Pereira, I. A.C. (2013). Unifying concepts in anaerobic respiration: Insights from dissimilatory sulfur metabolism. BBA- Bioenergetics. 145-160.
[5] Friedrich, C.G. Bardischewsky, F., Rother, D., Quentmeier, A., Fischer, J. (2005). Prokaryotic sulfur oxidation. Curr Opin Microbiol. 8, 253-259.
[6] Tourova, T.P., Slobodova, N.V., Bumazhkin, B.K., Kolganova, T.V., Muyzer, G., Sorokin, D.Y. (2012). Analysis of community composition of sulfur-oxidizing bacteria in hypersaline and soda lakes using soxB as a functional molecular marker. Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.
[7] Petri, R., Podgorsek, L., Imhoff, J.F. (2001). Phylogeny and distribution of the soxB gene among thiosulfate-oxidizing bacteria. FEMS Microbiology Letters. 197, 171-178.
1) Sample collection from a digester: • Anaerobic digested sludge samples containing SRB • Aerobic biofilm removed from concrete surface containing SOB
2) Isolation of genomic DNA from environmental samples: direct cell lysis with glass beads and DNA extraction using phenol chloroform method
3) Detection and identification of relevant SRB and SOB by PCR, PCR-DGGE, phylogenetic sequence analysis and FISH
4) Quantification of SRB and SOB using qPCR technique: selection of specific primer systems from literature data or as needed new primer design and evaluation of qPCR conditions.
SRB
(heterotrophic):
e.g. Desulfovibrio,
Desulfobacter
SOB
(autotrophic):
e.g. Thiobacillus,
Paracoccus
Anaerobic
biofilm
Aerobic
biofilm
Condensate
film
Abiotic corrosion Biotic corrosion
CO2 + H2O → H2CO3
H2S → H+ + HS-
H2S + 2O2 SOB H2SO4
Wastewater
Liquid-gas exchange
Sewer headspace
Figure 2. Severe BSA attack in a digester. © R. König
2 e-
AMP
Sat
APS
reductase
(ApsAB)
Dissimilatory sulfite
reductase
DsrAB/ DsrC
Sulfate
Adenosine 5´-phosphosulfate
(APS)
Sulfite
Sulfide
ATP
2Pi PPi
6 e-
Figure 3. Dissimilatory sulfate reduction [4].
• Dsr and aps genes (Figure 3) are present in all known sulfate reducing prokaryotes and have been proposed to be useful phylogenetic markers for SRB.
• Specific primers detecting dsrA and apsA genes have been used in a qPCR SybrGreen approach [3] and will be applied in the present study.
Functional molecular markers for SRB
• SOB can be detected and identified using the multi-enzyme Sox (sulfur-oxidizing) complex consisting of SoxB, SoxXA, SoxYZ and SoxCD components [5].
• Sox pathway is described in photo- and chemotrophic SOB that oxidize sulfide and thiosulfate to sulfate [6].
H2S + 2 O2 → SO42- + 2 H+
S2O32- + H2O + 2 O2 → 2 SO4
2- + 2 H+
• Primer system using the soxB gene which encodes the sulfate thiohydrolase, a key enzyme of the Sox pathway, has been applied in PCR (DNA fragment > 1000bp, [7]). For detecting soxB genes by qPCR a new approach has to be designed and evaluated.
Functional molecular markers for SOB
