Variable Variable- Pressure Scanning Electron Microscopy Pressure Scanning Electron Microscopy as experimental approach to studying as experimental approach to studying Biofilms Biofilms Lydia Lydia- Marie Joubert Marie Joubert 1 and Gideon Wolfaardt and Gideon Wolfaardt 2 1 Cell Sciences Imaging Facility, Stanford University, Stanford, U Cell Sciences Imaging Facility, Stanford University, Stanford, USA. SA. 2 Dept Chemistry and Biology, Ryerson University, Toronto, Canada. Dept Chemistry and Biology, Ryerson University, Toronto, Canada. Introduction: The importance of biofilms - i.e. microbial existence as attached communities, embedded in extracellular polymeric substances (EPS) - have profound medical, industrial and environmental implications. A major concern is their recalcitrance to antimicrobial agents, as well as to host defenses. Inclusion of microorganisms from various taxa, environmental niches and microbial lifestyles are furthermore necessitated by global issues on recombinant technology, biofuel production and xenobiotic degradation. Application and integration of multiple techniques facilitate a holistic approach to elucidate the biofilm phenomenon. Visualization techniques form a generic part of biofim studies, but with limited application of Electron Microscopy (EM). Biofilms EPS form a hydrogel, which renders this microbial phenomenon complex to visualize using normal EM preparative techniques. However, our application of Variable-Pressure Scanning Electron Microscopy (VP SEM) in various biofilm studies provided supporting evidence to data which is of major concern in industrial and natural environments. Methods: Biofilms from natural and lab-scale systems were viewed unfixed and hydrated with a LEO 1430 VP SEM after gold sputter-coating and application of conductive carbon paste. Operation at 7kV allowed high magnification imaging for periods of 30 min, with little evidence of structural distortion. Improved visualization of both cells and extracellular matrix (EPS) was allowed using the Variable Pressure system. VP SEM allows high-resolution, high magnification visualization under high pressure, thus tolerating a saturated chamber atmosphere, resulting in little deformation of hydrated biofilm structure. Such EM operation under humid conditions reduce the risk of reporting on artefacts in hydrated EPS with little structural integrity. Project 2: Role of attachment in the production of cellulolytic enzymes. Rationale : Enzymatic degradation of lignocellulose by the anaerobic rumen bacteria Ruminococcus albus and R.flavefaciens are commercially applied for ethanol production. Adherence to the substrate has been identified as imperative for cellulolytic enzyme production by the cellulosome. Limited knowledge exist on the mode of microbial attachment and character of the extracellular matrix for anaerobic cellulolysis. Experimental: R.albus7 and R.flavefaciens were grown on microcrystalline cellulose and cellobiose, and observed either as attached cells, or, after chemical extraction, as cellulose fibers with residual extracellular matrix, or degraded fibres without cells or EPS. Biochemical analyses of different fractions was applied. Integration Characterisation of the EPS, and visualization of microbial attachment revealed a close association between rumen bacteria and substrate, with different morphological forms on different substrates, and pili and glycocalyx formation apparent as modes of attachment LKA1 R. flavefaciens on cellulose R. flavefaciens on cellobiose R.albus7 : microbes, EPS & partially degraded cellulose Cells&EPS removed, cellulose remain Cells removed, EPS visible R. albus on cellobiose R.albus on cellulose Project 4: Role of flocculation in ethanol production by recombinant yeast strains Rationale: Bioconversion of starch to ethanol is enabled by by recombinant Saccharomyces cerevisiae strains expressing the raw starch-degrading α-amylase gene (LKA1) from Lipomyces kanonenkoae. Both flocculent and non-flocculent S. cerevisiae strains are applied, with flocculent strains (BELK1F) degrading starch with an earlier hydrolytic window than non-flocculent strains (BELK1), despite similar levels of α- amylase in the extracellular media – thereby decreasing the interval for enzyme-substrate interaction. Experimental: Flocculent strains (BELK1F) utilized 82% of raw starch, producing 5.1g/L ethanol – while non-flocculating strains (BELK1) consumed 76% starch, producing 4.61g/L ethanol. Integration: Fermentation studies integrated with VPSEM suggest that flocculation allows the secreted enzymes to act more efficiently on substrate degradation during fermentation. Physico-chemical properties like adhesion and hydrophobicity impact on biocatalyst performance. Flocculent BELK1F 0h 0h 0h 52h 36h 48h 24h 68h Glucose Maltose maltotriose Non-flocculent BELK1 0h 24h 48h 52h 68h 36h Glucose Maltose maltotriose Conclusions: In all of the above-mentioned projects VP SEM as visualization technique has proved to be of tremendous potential to be integrated with novel and existing techniques to enhance data retrieval. The challenge lies in integration of old with new techniques, and new with other novel approaches. Acknowledgments: The discussed research was incorporated into PhD projects of K du Plessis(3), H Schoeman(1) and N Ramachandran (4) (University Stellenbosch, SA), or resulted from cooperation with P Weimer (2)(UW Madison). The SEM facility, University Stellenbosch (SA) is thanked for technical support. Claude Leon Foundation (SA) is gratefully acknowleded for financial support for L-M Joubert. Linkage distance Linkage distance Linkage distance Complete Linkage Percent disagreement 11 10 9 8 5 7 6 4 3 2 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Linkage distance 0 100 200 300 400 500 600 0 1 2 3 4 5 6 7 8 9 10 11 week Fragment size week A B TRFLP data and cluster analysis of biofilm communities over time, including a COD shock at week 8 a b a b a b a b a b a b a b a b a b 0 100 200 300 400 500 600 9 a 9 b 10 a 10 b 11 a 11 b week a– Gravel inserted at initiation of experiment b – Gravel inserted after COD shock at week 8 Fragment size TRFLP data comparing community dynamics in established and new biofilms after COD shock Project 3: Wetland stability in treatment of wastewater Rationale: A currently explored low-cost method for the degradation of industrial effluent, as demanded by strict water quality legislation, is the application of constructed wetlands, where pollutant degradation and nutrient cycling are facilitated by microbial metabolic processes. Complexity of winery and distillery effluent necessitate strict control of wetland stability, with microbes, due to their short life cycles and rapid response to fluctuations, can be used as indicator of wetland health. As a survival mode under adverse growth conditions, biofilms act as stabilizer of microbial communities under fluctuating environmental conditions. Experimental : Biofilm and planktonic communities in experimental constructed wetlands were characterized and visualized, to be used as predictive and management tool for wetland stability. Gravel samples from various regions within the experimental wetlands were sampled periodically (incorporating a COD shock) to obtain both molecular and visual fingerprints of wetland microbial communities Integration: Visual data from VP SEM, integrated with molecular fingerprints from tRFLP, indicated extreme variation in biofilm communities of different regions of the wetland, influent and effluent, attached and planktonic communities of the same region, different soil types, as well as different retention times. Variation suggests adaptability, with microbial communities maintaining metabolic function through modifying species composition in response to fluctuation in their environment. Visual data indicated population distribution and biofilm architecture, EPS and stratification of biofilm layers, as well as spatial and temporal variation in biofilm structure. Biofilm dynamics can be deduced from structural variation - an aspect not afforded by tRFLP alone. Wk1 Wk2 Wk5 Wk6 Wk 7 Wk 8 Wk4 Wk3 LKA2 Cryptococcus LKA1 Saccharomyces Project 1: Environmental impact of GMO technology Rationale: Microbes present to us a vast array of usable products, but in limited amounts. Application of recombinant technology on selected superior strains increase fermentation productivity and reduce costs – but with limited insight into the survival of such genetically manipulated organisms in the environment. Experimental: Biofilm development by Cryptococcus laurentii and Saccharmoyces cerevisiae were compared to GMO strains LKA1 and LKA2 (S.cerevisiae strains expressing the raw starch-degrading genes encoding for α-amylases from Lipomyces kononenkoae Integration: Quantitative real-time monitoring of biofilm development, correlated with cell numbers in effluent, and integrated with VP-SEM, revealed diminished biofilm capacity of GMO strains, and less vigorous proliferation of recombinant yeasts
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Integration of Integration of VariableVariable--Pressure Scanning Electron MicroscopyPressure Scanning Electron Microscopy
as experimental approach to studying as experimental approach to studying BiofilmsBiofilmsLydiaLydia--Marie JoubertMarie Joubert11 and Gideon Wolfaardtand Gideon Wolfaardt22
11Cell Sciences Imaging Facility, Stanford University, Stanford, UCell Sciences Imaging Facility, Stanford University, Stanford, USA.SA.22Dept Chemistry and Biology, Ryerson University, Toronto, Canada.Dept Chemistry and Biology, Ryerson University, Toronto, Canada.
Introduction: The importance of biofilms - i.e. microbial existence as attached communities, embedded in extracellular polymeric substances (EPS) - have profound medical, industrial and environmental implications. A major concern is their recalcitrance to antimicrobial agents, as well as to host defenses. Inclusion of microorganisms from various taxa, environmental niches and microbial lifestyles are furthermore necessitated by global issues on recombinant technology, biofuel production and xenobiotic degradation. Application and integration of multiple techniques facilitate a holistic approach to elucidate the biofilm phenomenon. Visualization techniques form a generic part of biofimstudies, but with limited application of Electron Microscopy (EM). Biofilms EPS form a hydrogel, which renders this microbial phenomenon complex to visualize using normal EM preparative techniques. However, our application of Variable-Pressure Scanning Electron Microscopy (VP SEM) in various biofilm studies provided supporting evidence to data which is of major concern in industrial and natural environments.Methods: Biofilms from natural and lab-scale systems were viewed unfixed and hydrated with a LEO 1430 VP SEM after gold sputter-coating and application of conductive carbon paste. Operation at 7kV allowed high magnification imaging for periods of 30 min, with little evidence of structural distortion. Improved visualization of both cells and extracellular matrix (EPS) was allowed using the Variable Pressure system. VP SEM allows high-resolution, high magnification visualization under high pressure, thus tolerating a saturated chamber atmosphere, resulting in little deformation of hydrated biofilm structure. Such EM operation under humid conditions reduce the risk of reporting on artefacts in hydrated EPS with little structural integrity.
Project 2: Role of attachment in the production of cellulolytic enzymes.Rationale: Enzymatic degradation of lignocellulose by the anaerobic rumen bacteria Ruminococcusalbus and R.flavefaciens are commercially applied for ethanol production. Adherence to the substrate has been identified as imperative for cellulolytic enzyme production by the cellulosome. Limited knowledge exist on the mode of microbial attachment and character of the extracellular matrix for anaerobic cellulolysis. Experimental: R.albus7 and R.flavefaciens were grown on microcrystalline cellulose and cellobiose, and observed either as attached cells, or, after chemical extraction, as cellulose fibers with residual extracellular matrix, or degraded fibres without cells or EPS. Biochemical analyses of different fractions was applied. Integration Characterisation of the EPS, and visualization of microbial attachment revealed a close association between rumen bacteria and substrate, with different morphological forms on different
substrates, and pili and glycocalyx formation apparent as modes of attachment
LKA1
R. flavefaciens on cellulose R. flavefaciens on cellobiose
R.albus7 : microbes, EPS & partially degraded cellulose
Cells&EPS removed, cellulose remainCells removed, EPS visible
R. albus on cellobioseR.albus on cellulose
Project 4: Role of flocculation in ethanol production by recombinant yeast strainsRationale: Bioconversion of starch to ethanol is enabled by by recombinant Saccharomyces cerevisiaestrains expressing the raw starch-degrading α-amylase gene (LKA1) from Lipomyces kanonenkoae. Both flocculent and non-flocculent S. cerevisiae strains are applied, with flocculent strains (BELK1F) degrading starch with an earlier hydrolytic window than non-flocculent strains (BELK1), despite similar levels of α-amylase in the extracellular media – thereby decreasing the interval for enzyme-substrate interaction.Experimental: Flocculent strains (BELK1F) utilized 82% of raw starch, producing 5.1g/L ethanol – while non-flocculating strains (BELK1) consumed 76% starch, producing 4.61g/L ethanol. Integration: Fermentation studies integrated with VPSEM suggest that flocculation allows the secreted enzymes to act more efficiently on substrate degradation during fermentation. Physico-chemical properties like adhesion and hydrophobicity impact on biocatalyst performance.
Flocculent BELK1F
0h 0h
0h
52h
36h
48h
24h
68h
GlucoseMaltosemaltotriose
Non-flocculent BELK10h 24h
48h 52h 68h
36h
GlucoseMaltosemaltotriose
Conclusions: In all of the above-mentioned projects VP SEM as visualization technique has proved to be of tremendous potential to be integrated with novel and existing techniques to enhance data retrieval. The challenge lies in integration of old with new techniques, and new with other novel approaches.
Acknowledgments: The discussed research was incorporated into PhD projects of K du Plessis(3), H Schoeman(1) and N Ramachandran (4) (University Stellenbosch, SA), or resulted from cooperation with P Weimer (2)(UW Madison). The SEM facility, University Stellenbosch (SA) is thanked for technical support. Claude Leon Foundation (SA) is gratefully acknowleded for financial support for L-M Joubert.
Complete LinkagePercent disagreement
11 10 9 8 5 7 6 4 3 20.0
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Link
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Complete LinkagePercent disagreement
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11 10 9 8 5 7 6 4 3 20.0
0.1
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0.6
Link
age
dist
ance
Complete LinkagePercent disagreement
11 10 9 8 5 7 6 4 3 20.0
0.1
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0.6
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dist
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weekA B
TRFLP data and cluster analysis
of biofilmcommunities
over time, including a COD shock at week 8
0
100
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9a 9 b 10 a 10 b 11a 11b
week
a – Gravel inserted at initiation of experimentb – Gravel inserted after COD shock at week 8
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a – Gravel inserted at initiation of experimentb – Gravel inserted after COD shock at week 8
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a – Gravel inserted at initiation of experimentb – Gravel inserted after COD shock at week 8
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a – Gravel inserted at initiation of experimentb – Gravel inserted after COD shock at week 8
Frag
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TRFLP data comparing community dynamics in established and new biofilms after COD shock
Project 3: Wetland stability in treatment of wastewaterRationale: A currently explored low-cost method for the degradation of industrial effluent, as demanded by strict water quality legislation, is the application of constructed wetlands, where pollutant degradation and nutrient cycling are facilitated by microbial metabolic processes. Complexity of winery and distillery effluent necessitate strict control of wetland stability, with microbes, due to their short life cycles and rapid response to fluctuations, can be used as indicator of wetland health. As a survival mode under adverse growth conditions, biofilms act as stabilizer of microbial communities under fluctuating environmental conditions. Experimental: Biofilm and planktonic communities in experimental constructed wetlands were characterized and visualized, to be used as predictive and management tool for wetland stability. Gravel samples from various regions within the experimental wetlands were sampled periodically (incorporating a COD shock) to obtain both molecular and visual fingerprints of wetland microbial communitiesIntegration: Visual data from VP SEM, integrated with molecular fingerprints from tRFLP, indicated extreme variation in biofilm communities of different regions of the wetland, influent and effluent, attached and planktonic communities of the same region, different soil types, as well as different retention times.Variation suggests adaptability, with microbial communities maintaining metabolic function through modifying species composition in response to fluctuation in their environment. Visual data indicated population distribution and biofilm architecture, EPS and stratification of biofilm layers, as well as spatial and temporal variation in biofilm structure. Biofilm dynamics can be deduced from structural variation -an aspect not afforded by tRFLP alone.
Wk1 Wk2
Wk5 Wk6 Wk 7 Wk 8
Wk4Wk3
LKA2Cryptococcus LKA1Saccharomyces
Project 1: Environmental impact of GMO technologyRationale: Microbes present to us a vast array of usable products, but in limited amounts. Application of recombinant technology on selected superior strains increase fermentation productivity and reduce costs – but with limited insight into the survival of such genetically manipulated organisms in the environment. Experimental: Biofilm development by Cryptococcus laurentii and Saccharmoyces cerevisiae were compared to GMO strains LKA1 and LKA2 (S.cerevisiae strains expressing the raw starch-degrading genes encoding for α-amylases from Lipomyces kononenkoaeIntegration: Quantitative real-time monitoring of biofilm development, correlated with cell numbers in effluent, and integrated with VP-SEM, revealed diminished biofilm capacity of GMO strains, and less vigorous proliferation of recombinant yeasts
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