1 univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slo... more 1 univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Assist., e-mail: gasan.osojnik@bfro.uni-lj.si 2 Same address, prof., pd.D., m.Sc., e-mail: romana.marinsek@bfro.uni-lj.si Identification of environmental impact hot spots in traditional food production lines Evaluating the environmental impact of agricultural food production systems is a relatively recent research activity and the present methods for assessing the environmental impact associated with production-consumption systems are inputoutput accounting, ecological footprint analysis, life cycle assessment, financial evaluation of environmental externalities, farm cost and food miles. Complete environmental impact analyses require considerable amounts of data and time and are very likely to be expensive. preliminary identification of the most-evident environmental hot spots is beneficial in aiding the determination of the analysis scope and goal and in setting the borders of t...
Degradation of brewery spent grain as a novel test substrate was explored in routine biochemical ... more Degradation of brewery spent grain as a novel test substrate was explored in routine biochemical methane potential assays (BMP) using three different inocula. Significant differences in the initial biogas production rates from spent grain, methane yield coefficients and final spent grain degradation were observed between inocula. Initial and developed communities degrading novel substrate showed significant differences in archaeal community fingerprints. Differences were observed irrespective of substrate identity (no substrate, glucose, spent grain) providing evidence of a significant general influence of BMP incubation on the microbial phylotypes. A linear relationship between microbial community flexibility in BMP assay and corresponding initial biogas production rates was identified as a novel parameter to diagnose anaerobic processes, particularly under dynamic conditions like start-up.
Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. C... more Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose. The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel. This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.
Lignocellulosic substrates are widely available but not easily applied in biogas production due t... more Lignocellulosic substrates are widely available but not easily applied in biogas production due to their poor anaerobic degradation. The effect of bioaugmentation by anaerobic hydrolytic bacteria on biogas production was determined by the biochemical methane potential assay. Microbial biomass from full scale upflow anaerobic sludge blanket reactor treating brewery wastewater was a source of active microorganisms and brewery spent grain a model lignocellulosic substrate. Ruminococcus flavefaciens 007C, Pseudobutyrivibrio xylanivorans Mz5T, Fibrobacter succinogenes S85 and Clostridium cellulovorans as pure and mixed cultures were used to enhance the lignocellulose degradation and elevate the biogas production. P. xylanivorans Mz5T was the most successful in elevating methane production (+ 17.8 %), followed by the coculture of P. xylanivorans Mz5T and F. succinogenes S85 (+ 6.9 %) and the coculture of C. cellulovorans and F. succinogenes S85 (+ 4.9 %). Changes in microbial community structure were detected by fingerprinting techniques.
In this study anaerobic digestion of selected lignocellulosic substrate, namely brewery spent gra... more In this study anaerobic digestion of selected lignocellulosic substrate, namely brewery spent grain (BSG), was studied. In order to facilitate anaerobic digestion several types of pretreatment methods were tested such as: mechanical, chemical (alkali and acid) and thermo-chemical. The anaerobic digestion experiments were carried out in a semi-continuous stirred bioreactors with the organic loading rates between 2.9 and 3.9 kgCOD m-3 d-1 (1.9 and 2.5 kgVSS m-3 d-1 respectively) and corresponding hydraulic retention times of 33-39 days. Biogas production and composition, pH, COD, TSS and VSS, short chain fatty acids and phenolic compounds were measured. A significant inhibition of biogas production occurred, depending on the type of substrate pretreatment. There are indications that p-cresol is responsible for process inhibition when its concentration in the reaction mixture exceeds critical value between 115 and 240 mg L-1. Anaerobic digestion of chemically pretreated BSG (acid and alkali) and untreated-raw BSG was inhibited between the days 56 and 63 of the experiment, followed by thermo-chemically pretreated BSG on day 112 and mechanically pretreated BSG on day 126. Analyses of the substrates showed no phenolic compounds either in raw-untreated BSG or pretreated substrates, therefore the recorded p-cresol is an intermediate degradation product, responsible for process inhibition.
Successful biogas production is based on stable or adaptable microbial community structure and ac... more Successful biogas production is based on stable or adaptable microbial community structure and activity which depends on type of substrate used and several physico-chemical conditions in the bioreactor. Monitoring those and the dynamics of microbiota is important for planning and optimizing the biogas process, avoiding critical points and reaching the maximum methane yield. Methanogens are extremely difficult to study with culture-based methods. Molecular methods for microbial community structure analysis in biogas reactors, which offer qualitative and quantitative information on bacterial and archaeal species and their microbial community changes, and causes for process instability are surveyed in this review. For comparative studies semi-quantitative, rapid and cheap techniques like T-RFLP, DGGE and TGGE are used. More laborious and expensive techniques with high-throughput like semi-quantitative FISH and DNA microarrays and also quantitative techniques like qPCR and sequencing are used for phylogenetic analysis. Technique type adequacy for certain study depends on what information is needed and on several advantages and disadvantages every technique possesses.
A successful biogas production process
depends upon adequate hydrolysis of macromolecules in
the ... more A successful biogas production process depends upon adequate hydrolysis of macromolecules in the substrate and stable further conversion. The complex and rigid structure of cellulosic, hemicellulosic and lignin chain is preventing lignocellulosic biomass to reach efficient hydrolysis, therefore pretreatment of a substrate is needed for higher biogas and methane yields. There are several different physical and chemical methods of pretreatments available which include the usage of acids, alkalis, organic solvents, ionic detergents, steam, high pressure, grinding, ultrasound, and microwave irradiation. Physico-chemical pretreatments act rapidly on lignocellulose but their upscaling is very expensive in industry. Many studies have been made in finding the best combination of different pretreatment methods and also new biological techniques which could make lignocellulose pretreatment cheaper and environmentally more friendly. Using natural abilities of different fungi, bacteria or yeast to degrade lignocellulose simplifies the whole process. Also cocktails of biotechnologically produced enzymes are effective in degrading lignocellulose.
1 univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slo... more 1 univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Assist., e-mail: gasan.osojnik@bfro.uni-lj.si 2 Same address, prof., pd.D., m.Sc., e-mail: romana.marinsek@bfro.uni-lj.si Identification of environmental impact hot spots in traditional food production lines Evaluating the environmental impact of agricultural food production systems is a relatively recent research activity and the present methods for assessing the environmental impact associated with production-consumption systems are inputoutput accounting, ecological footprint analysis, life cycle assessment, financial evaluation of environmental externalities, farm cost and food miles. Complete environmental impact analyses require considerable amounts of data and time and are very likely to be expensive. preliminary identification of the most-evident environmental hot spots is beneficial in aiding the determination of the analysis scope and goal and in setting the borders of t...
Degradation of brewery spent grain as a novel test substrate was explored in routine biochemical ... more Degradation of brewery spent grain as a novel test substrate was explored in routine biochemical methane potential assays (BMP) using three different inocula. Significant differences in the initial biogas production rates from spent grain, methane yield coefficients and final spent grain degradation were observed between inocula. Initial and developed communities degrading novel substrate showed significant differences in archaeal community fingerprints. Differences were observed irrespective of substrate identity (no substrate, glucose, spent grain) providing evidence of a significant general influence of BMP incubation on the microbial phylotypes. A linear relationship between microbial community flexibility in BMP assay and corresponding initial biogas production rates was identified as a novel parameter to diagnose anaerobic processes, particularly under dynamic conditions like start-up.
Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. C... more Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose. The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel. This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.
Lignocellulosic substrates are widely available but not easily applied in biogas production due t... more Lignocellulosic substrates are widely available but not easily applied in biogas production due to their poor anaerobic degradation. The effect of bioaugmentation by anaerobic hydrolytic bacteria on biogas production was determined by the biochemical methane potential assay. Microbial biomass from full scale upflow anaerobic sludge blanket reactor treating brewery wastewater was a source of active microorganisms and brewery spent grain a model lignocellulosic substrate. Ruminococcus flavefaciens 007C, Pseudobutyrivibrio xylanivorans Mz5T, Fibrobacter succinogenes S85 and Clostridium cellulovorans as pure and mixed cultures were used to enhance the lignocellulose degradation and elevate the biogas production. P. xylanivorans Mz5T was the most successful in elevating methane production (+ 17.8 %), followed by the coculture of P. xylanivorans Mz5T and F. succinogenes S85 (+ 6.9 %) and the coculture of C. cellulovorans and F. succinogenes S85 (+ 4.9 %). Changes in microbial community structure were detected by fingerprinting techniques.
In this study anaerobic digestion of selected lignocellulosic substrate, namely brewery spent gra... more In this study anaerobic digestion of selected lignocellulosic substrate, namely brewery spent grain (BSG), was studied. In order to facilitate anaerobic digestion several types of pretreatment methods were tested such as: mechanical, chemical (alkali and acid) and thermo-chemical. The anaerobic digestion experiments were carried out in a semi-continuous stirred bioreactors with the organic loading rates between 2.9 and 3.9 kgCOD m-3 d-1 (1.9 and 2.5 kgVSS m-3 d-1 respectively) and corresponding hydraulic retention times of 33-39 days. Biogas production and composition, pH, COD, TSS and VSS, short chain fatty acids and phenolic compounds were measured. A significant inhibition of biogas production occurred, depending on the type of substrate pretreatment. There are indications that p-cresol is responsible for process inhibition when its concentration in the reaction mixture exceeds critical value between 115 and 240 mg L-1. Anaerobic digestion of chemically pretreated BSG (acid and alkali) and untreated-raw BSG was inhibited between the days 56 and 63 of the experiment, followed by thermo-chemically pretreated BSG on day 112 and mechanically pretreated BSG on day 126. Analyses of the substrates showed no phenolic compounds either in raw-untreated BSG or pretreated substrates, therefore the recorded p-cresol is an intermediate degradation product, responsible for process inhibition.
Successful biogas production is based on stable or adaptable microbial community structure and ac... more Successful biogas production is based on stable or adaptable microbial community structure and activity which depends on type of substrate used and several physico-chemical conditions in the bioreactor. Monitoring those and the dynamics of microbiota is important for planning and optimizing the biogas process, avoiding critical points and reaching the maximum methane yield. Methanogens are extremely difficult to study with culture-based methods. Molecular methods for microbial community structure analysis in biogas reactors, which offer qualitative and quantitative information on bacterial and archaeal species and their microbial community changes, and causes for process instability are surveyed in this review. For comparative studies semi-quantitative, rapid and cheap techniques like T-RFLP, DGGE and TGGE are used. More laborious and expensive techniques with high-throughput like semi-quantitative FISH and DNA microarrays and also quantitative techniques like qPCR and sequencing are used for phylogenetic analysis. Technique type adequacy for certain study depends on what information is needed and on several advantages and disadvantages every technique possesses.
A successful biogas production process
depends upon adequate hydrolysis of macromolecules in
the ... more A successful biogas production process depends upon adequate hydrolysis of macromolecules in the substrate and stable further conversion. The complex and rigid structure of cellulosic, hemicellulosic and lignin chain is preventing lignocellulosic biomass to reach efficient hydrolysis, therefore pretreatment of a substrate is needed for higher biogas and methane yields. There are several different physical and chemical methods of pretreatments available which include the usage of acids, alkalis, organic solvents, ionic detergents, steam, high pressure, grinding, ultrasound, and microwave irradiation. Physico-chemical pretreatments act rapidly on lignocellulose but their upscaling is very expensive in industry. Many studies have been made in finding the best combination of different pretreatment methods and also new biological techniques which could make lignocellulose pretreatment cheaper and environmentally more friendly. Using natural abilities of different fungi, bacteria or yeast to degrade lignocellulose simplifies the whole process. Also cocktails of biotechnologically produced enzymes are effective in degrading lignocellulose.
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on type of substrate used and several physico-chemical conditions in the bioreactor. Monitoring those and the dynamics
of microbiota is important for planning and optimizing the biogas process, avoiding critical points and reaching the maximum
methane yield. Methanogens are extremely difficult to study with culture-based methods. Molecular methods
for microbial community structure analysis in biogas reactors, which offer qualitative and quantitative information on
bacterial and archaeal species and their microbial community changes, and causes for process instability are surveyed in
this review. For comparative studies semi-quantitative, rapid and cheap techniques like T-RFLP, DGGE and TGGE are
used. More laborious and expensive techniques with high-throughput like semi-quantitative FISH and DNA microarrays
and also quantitative techniques like qPCR and sequencing are used for phylogenetic analysis. Technique type adequacy
for certain study depends on what information is needed and on several advantages and disadvantages every technique
possesses.
depends upon adequate hydrolysis of macromolecules in
the substrate and stable further conversion. The complex
and rigid structure of cellulosic, hemicellulosic and lignin
chain is preventing lignocellulosic biomass to reach efficient
hydrolysis, therefore pretreatment of a substrate is
needed for higher biogas and methane yields. There are
several different physical and chemical methods of pretreatments
available which include the usage of acids,
alkalis, organic solvents, ionic detergents, steam, high
pressure, grinding, ultrasound, and microwave irradiation.
Physico-chemical pretreatments act rapidly on lignocellulose
but their upscaling is very expensive in industry. Many
studies have been made in finding the best combination of
different pretreatment methods and also new biological
techniques which could make lignocellulose pretreatment
cheaper and environmentally more friendly. Using natural
abilities of different fungi, bacteria or yeast to degrade
lignocellulose simplifies the whole process. Also cocktails
of biotechnologically produced enzymes are effective in
degrading lignocellulose.
on type of substrate used and several physico-chemical conditions in the bioreactor. Monitoring those and the dynamics
of microbiota is important for planning and optimizing the biogas process, avoiding critical points and reaching the maximum
methane yield. Methanogens are extremely difficult to study with culture-based methods. Molecular methods
for microbial community structure analysis in biogas reactors, which offer qualitative and quantitative information on
bacterial and archaeal species and their microbial community changes, and causes for process instability are surveyed in
this review. For comparative studies semi-quantitative, rapid and cheap techniques like T-RFLP, DGGE and TGGE are
used. More laborious and expensive techniques with high-throughput like semi-quantitative FISH and DNA microarrays
and also quantitative techniques like qPCR and sequencing are used for phylogenetic analysis. Technique type adequacy
for certain study depends on what information is needed and on several advantages and disadvantages every technique
possesses.
depends upon adequate hydrolysis of macromolecules in
the substrate and stable further conversion. The complex
and rigid structure of cellulosic, hemicellulosic and lignin
chain is preventing lignocellulosic biomass to reach efficient
hydrolysis, therefore pretreatment of a substrate is
needed for higher biogas and methane yields. There are
several different physical and chemical methods of pretreatments
available which include the usage of acids,
alkalis, organic solvents, ionic detergents, steam, high
pressure, grinding, ultrasound, and microwave irradiation.
Physico-chemical pretreatments act rapidly on lignocellulose
but their upscaling is very expensive in industry. Many
studies have been made in finding the best combination of
different pretreatment methods and also new biological
techniques which could make lignocellulose pretreatment
cheaper and environmentally more friendly. Using natural
abilities of different fungi, bacteria or yeast to degrade
lignocellulose simplifies the whole process. Also cocktails
of biotechnologically produced enzymes are effective in
degrading lignocellulose.