Biochemical characterization of the β-mannanase activity of Bacillus paralicheniformis SVD1
- Authors: Clarke, Matthew David
- Date: 2019
- Subjects: Mycobacterium avium paratuberculosis , Enzymes -- Biotechnology , Lignocellulose -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67570 , vital:29112
- Description: Products produced via the enzymatic hydrolysis of lignocellulosic biomass, the most abundant renewable terrestrial source of carbon, can potentially replace a lot of the fuels and chemicals currently produced using non-renewable hydrocarbons. Mannan is a polysaccharide component of lignocellulose that is abundant in softwoods and legume seeds. Enzymatic hydrolysis of mannan by β-mannanases has various industrial applications, including use in biofuel and prebiotic mannooligosaccharide (MOS) production for the improvement of human and animal health. The industrial use of β-mannanases depends on their biochemical characteristics, such as their activity, stability and substrate specificity. Knowledge of their synergistic interactions with other enzymes is also useful for effective hydrolysis. Bacillus paralicheniformis SVD1 was used as a source for β-mannanases. The two mannanases of B. paralicheniformis SVD1 have not been biochemically characterized apart from minor characterization of crude β-mannanase activity. The protein sequences of the two β-mannanases, of glycosyl hydrolase family 5 and 26, have a 95% - 96% identity to the β-mannanases of B. licheniformis DSM13T (=ATCC14580T). These small protein sequence differences could lead to quite different biochemical characteristics. These mannanases were characterized as these enzymes may have industrially useful characteristics. To induce mannanase production, B. paralicheniformis SVD1 was cultured in broth containing the mannan substrate locust bean gum. Various growth curve parameters were measured over 72 h. Mannanase activity was the highest after 48 h of growth - this was the time at which mannanase activity was concentrated, using 3 kDa centrifugal filtration devices, for biochemical characterization of the crude activity. Zymography revealed that the crude concentrated mannanase fraction consisted of at least two mannanases with relative molecular weights (MWs) of 29.6 kDa and 33 kDa. This was smaller than expected – based on their theoretical molecular masses. Protease activity, which was detected in the broth, was probably the reason. There were two pH optima, pH 5.0 and pH 7.0, which also indicated the presence of two mannanases. The concentrated mannanase displayed characteristics that were expected of a B. paralicheniformis β-mannanase. The temperature optimum was 50°C and the activity loss was less than 7% at 50°C after 24 h. Substrate specificity assays revealed that there was predominantly mannanase activity present. Thin layer chromatography (TLC) analysis of mannan and MOS hydrolysis showed that mainly M2 and M3 MOS were produced; only MOS with a degree of polymerization of 4 or higher were hydrolyzed. Hydrolysis was minimal on mannoligosaccharides with galactose substituents. Activity and MOS production was the highest on soluble, low branched mannan substrates. The highest activity observed was on konjac glucomannan. Purification of the mannanase activity was then attempted using various methods. Ammonium sulfate precipitation, acetone precipitation, as well as centrifugal filtration device concentration was assessed for concentration of the mannanase activity.Concentration was not very successful due to low activity yields (≤ 20%). Anion exchange chromatography (AEC) and size exclusion chromatography (SEC) was used for purification. AEC gave good activity yield and fold purification, but SDS-PAGE analysis revealed the presence of many different proteins so further purification was necessary. SDS-PAGE analysis showed that there were only a few protein contaminants in the SEC fraction. However, the yield was too low to allow for biochemical characterization. The optimized purification procedure, which partially purified the mannanase activity, used 85% ammonium sulfate precipitation, followed by AEC. The fold purification was high (88.9) and the specific activity was 29.5 U.mg-1. A zymogram of the partially purified mannanase showed a mannanase active band with a MW of 40 - 41 kDa. A serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was added during the purification steps. This indicated that the mannanase/s in the crude concentrate, without PMSF added, was hydrolyzed by serine protease activity. Native PAGE zymograms suggested that at least two different isoforms of mannanases were present. Additional purification would be required to determine the true characteristics of the mannanase/s. The biochemical characteristics of the crude and partially purified mannanases were similar. The pH optima of the partially purified mannanases were different; the pH optima were 6.0 and 9.0. The substrate specificities were similar, except that the partially purified mannanases displayed no cellulase and β-D-galactosidase activity, but showed a small amount of α-L-arabinase activity. The partially purified mannanase and a Cyamopsis tetragonolobus GH27 α-galactosidase synergistically hydrolyzed locust bean gum. The M50G50 combination displayed the highest extent of hydrolysis; after 24 h there was a 1.39 fold increase in reducing sugar release and the degree of synergy (DS) was 4.64. TLC analysis indicated that synergy increased the release of small MOS. These MOS could be useful as prebiotics. The synergy between the partially purified mannanase and the commercial cellulase mixture Cellic® CTec2 (Novozymes) on spent coffee grounds (SCG) was also determined. SCG is an abundant industrial waste product that has high mannan content. The SCG was pretreated using NaOH, and the monosaccharide, soluble phenolics and insoluble contents were determined. Glucose and mannose were the dominant monosaccharides in the SCG; the pretreated SCG contained 20.4% (w/w) glucose and 18.5% (w/w) mannose, respectively. The NaOH pretreatment improved mannanase hydrolysis of SCG. It resulted in the opening up and swelling of the SCG particles and removed some of the insoluble solids. The partially purified B. paralicheniformis SVD1 mannanase displayed no detectable activity on SCG, but showed synergy with CTec2, in terms of DS, on untreated and NaOH pretreated SCG. This is the first report of mannanasecellulase synergy on SCG; other studies found that increased hydrolysis was due to additive effects. The results obtained in this study are only an initial assessment of the biochemical properties of B. paralicheniformis SVD1 mannanase activity and its synergy with other enzymes. These results can be used to inform future studies.
- Full Text:
- Date Issued: 2019
- Authors: Clarke, Matthew David
- Date: 2019
- Subjects: Mycobacterium avium paratuberculosis , Enzymes -- Biotechnology , Lignocellulose -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67570 , vital:29112
- Description: Products produced via the enzymatic hydrolysis of lignocellulosic biomass, the most abundant renewable terrestrial source of carbon, can potentially replace a lot of the fuels and chemicals currently produced using non-renewable hydrocarbons. Mannan is a polysaccharide component of lignocellulose that is abundant in softwoods and legume seeds. Enzymatic hydrolysis of mannan by β-mannanases has various industrial applications, including use in biofuel and prebiotic mannooligosaccharide (MOS) production for the improvement of human and animal health. The industrial use of β-mannanases depends on their biochemical characteristics, such as their activity, stability and substrate specificity. Knowledge of their synergistic interactions with other enzymes is also useful for effective hydrolysis. Bacillus paralicheniformis SVD1 was used as a source for β-mannanases. The two mannanases of B. paralicheniformis SVD1 have not been biochemically characterized apart from minor characterization of crude β-mannanase activity. The protein sequences of the two β-mannanases, of glycosyl hydrolase family 5 and 26, have a 95% - 96% identity to the β-mannanases of B. licheniformis DSM13T (=ATCC14580T). These small protein sequence differences could lead to quite different biochemical characteristics. These mannanases were characterized as these enzymes may have industrially useful characteristics. To induce mannanase production, B. paralicheniformis SVD1 was cultured in broth containing the mannan substrate locust bean gum. Various growth curve parameters were measured over 72 h. Mannanase activity was the highest after 48 h of growth - this was the time at which mannanase activity was concentrated, using 3 kDa centrifugal filtration devices, for biochemical characterization of the crude activity. Zymography revealed that the crude concentrated mannanase fraction consisted of at least two mannanases with relative molecular weights (MWs) of 29.6 kDa and 33 kDa. This was smaller than expected – based on their theoretical molecular masses. Protease activity, which was detected in the broth, was probably the reason. There were two pH optima, pH 5.0 and pH 7.0, which also indicated the presence of two mannanases. The concentrated mannanase displayed characteristics that were expected of a B. paralicheniformis β-mannanase. The temperature optimum was 50°C and the activity loss was less than 7% at 50°C after 24 h. Substrate specificity assays revealed that there was predominantly mannanase activity present. Thin layer chromatography (TLC) analysis of mannan and MOS hydrolysis showed that mainly M2 and M3 MOS were produced; only MOS with a degree of polymerization of 4 or higher were hydrolyzed. Hydrolysis was minimal on mannoligosaccharides with galactose substituents. Activity and MOS production was the highest on soluble, low branched mannan substrates. The highest activity observed was on konjac glucomannan. Purification of the mannanase activity was then attempted using various methods. Ammonium sulfate precipitation, acetone precipitation, as well as centrifugal filtration device concentration was assessed for concentration of the mannanase activity.Concentration was not very successful due to low activity yields (≤ 20%). Anion exchange chromatography (AEC) and size exclusion chromatography (SEC) was used for purification. AEC gave good activity yield and fold purification, but SDS-PAGE analysis revealed the presence of many different proteins so further purification was necessary. SDS-PAGE analysis showed that there were only a few protein contaminants in the SEC fraction. However, the yield was too low to allow for biochemical characterization. The optimized purification procedure, which partially purified the mannanase activity, used 85% ammonium sulfate precipitation, followed by AEC. The fold purification was high (88.9) and the specific activity was 29.5 U.mg-1. A zymogram of the partially purified mannanase showed a mannanase active band with a MW of 40 - 41 kDa. A serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was added during the purification steps. This indicated that the mannanase/s in the crude concentrate, without PMSF added, was hydrolyzed by serine protease activity. Native PAGE zymograms suggested that at least two different isoforms of mannanases were present. Additional purification would be required to determine the true characteristics of the mannanase/s. The biochemical characteristics of the crude and partially purified mannanases were similar. The pH optima of the partially purified mannanases were different; the pH optima were 6.0 and 9.0. The substrate specificities were similar, except that the partially purified mannanases displayed no cellulase and β-D-galactosidase activity, but showed a small amount of α-L-arabinase activity. The partially purified mannanase and a Cyamopsis tetragonolobus GH27 α-galactosidase synergistically hydrolyzed locust bean gum. The M50G50 combination displayed the highest extent of hydrolysis; after 24 h there was a 1.39 fold increase in reducing sugar release and the degree of synergy (DS) was 4.64. TLC analysis indicated that synergy increased the release of small MOS. These MOS could be useful as prebiotics. The synergy between the partially purified mannanase and the commercial cellulase mixture Cellic® CTec2 (Novozymes) on spent coffee grounds (SCG) was also determined. SCG is an abundant industrial waste product that has high mannan content. The SCG was pretreated using NaOH, and the monosaccharide, soluble phenolics and insoluble contents were determined. Glucose and mannose were the dominant monosaccharides in the SCG; the pretreated SCG contained 20.4% (w/w) glucose and 18.5% (w/w) mannose, respectively. The NaOH pretreatment improved mannanase hydrolysis of SCG. It resulted in the opening up and swelling of the SCG particles and removed some of the insoluble solids. The partially purified B. paralicheniformis SVD1 mannanase displayed no detectable activity on SCG, but showed synergy with CTec2, in terms of DS, on untreated and NaOH pretreated SCG. This is the first report of mannanasecellulase synergy on SCG; other studies found that increased hydrolysis was due to additive effects. The results obtained in this study are only an initial assessment of the biochemical properties of B. paralicheniformis SVD1 mannanase activity and its synergy with other enzymes. These results can be used to inform future studies.
- Full Text:
- Date Issued: 2019
Chitin hydrolysis with chitinolytic enzymes for the production of chitooligomers with antimicrobial properties
- Authors: Oree, Glynis
- Date: 2019
- Subjects: Chitin -- Biotechnology , Enzymes -- Biotechnology , Hydrolysis , Chitooligomers -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67887 , vital:29165
- Description: There are many diseases and illnesses in the world that require new drug treatments and chitin has been shown to produce chitooligomeric derivatives which exhibit promising antimicrobial and immune-enhancing properties. However, the rate-limiting step is associated with the high recalcitrance of chitinous substrates, and low hydrolytic activities of chitinolytic enzymes, resulting in low product release. To improve and create a more sustainable and economical process, enhancing chitin hydrolysis through various treatment procedures is essential for obtaining high enzyme hydrolysis rates, resulting in a higher yield of chitooligomers (CHOS). In literature, pre-treatment of insoluble biomass is generally associated with an increase in accessibility of the carbohydrate to hydrolytic enzymes, thus generating more products. The first part of this study investigated the effect of alkali- (NaOH) and acid pre-treatments (HCl and phosphoric acid) on chitin biomass, and chemical and morphological modifications were assessed by the employment of scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Energy-Dispersive X-ray spectrometery (EDX) and x-ray diffraction (XRD). Data obtained confirmed that pre-treated substrates were more chemically and morphologically modified. These results confirmed the fact that pre-treatment of chitin disrupts the structure of the biomass, rendering the polymer more accessible for enzymatic hydrolysis. The commercial chitinases from Bacillus cereus and Streptomyces griseus (CHB and CHS) are costly. Bio-prospecting for other chitin-degrading enzymes from alternate sources such as Oidiodendron maius, or the recombinant expression of CHOS, was a more economically feasible avenue. The chit1 gene from Thermomyces lanuginosus, expressed in Pichia pastoris, produced a large range CHOS with a degree of polymerisation (DP) ranging from 1 to above 6. TLC analysis showed that O. maius exhibited chitin-degrading properties by producing CHOS with a DP length of 1 to 3. These two sources were therefore successful in producing chitin-degrading enzymes. The physico-chemical properties of commercial (CHB and CHS) and expressed (Chit1) chitinolytic enzymes were investigated, to determine under which biochemical conditions and on which type of biomass they can function on optimally, for the production of value-added products such as CHOS. Substrate affinity assays were conducted on the un-treated and pre-treated biomass. TLC revealed that chitosan hydrolysis by the commercial chitinases produced the largest range of CHOS with a DP length ranging from 1 to 6. A range of temperatures (35-90oC) were investigated and CHB, CHS and Chit1 displayed optimum activities at 50, 40 and 45 oC, respectively. Thermostability studies that were conducted at 37 and 50oC revealed that CHB and CHS were most stable at 37oC. Chit1 showed great thermostablity at both temperatures, rendering this enzyme suitable for industrial processes at high temperatures. pH optima studies demonstrated that the pH optima for CHB, CHS and Chit1 was at a pH of 5.0, with specific activities of 33.459, 46.2 and 5.776 μmol/h/mg, respectively. The chain cleaving patterns of the commercial enzymes were determined and exo-chitinase activity was exhibited, due to the production of CHOS that were predominantly of a DP length of 2. Enzyme binary synergy studies were conducted with commercial chitinases (CHB and CHS) on colloidal chitin. Studies illustrated that the simultaneous combination of CHB 75%: CHS 25% produced the highest specific activity (3.526 μmol/h/mg), with no synergy. TLC analysis of this enzyme combination over time revealed that predominantly chitobiose was produced. This suggested that the substrate crystallinity and morphology played an important role in the way the enzymes cleaved the carbohydrate. Since CHOS have shown great promise for their antimicrobial properties, the CHOS generated from the chitinous substrates were tested for antimicrobial properties on Bacillus subtilis, Escherichia coli, Klebsiella and Staphlococcus aureus. This study revealed that certain CHOS produced have inhibitory effects on certain bacteria and could potentially be used in the pharamceutical or medical industries. In conclusion, this study revealed that chitinases can be produced and found in alternate sources and be used for the hydrolysis of chitinous biomass in a more sustainabe and economically viable manner. The chitinases investigated (CHB, CHS and Chit1) exhibited different cleaving patterns of the chitinous substrates due to the chemical and morphological properties of the biomass. CHOS produced from chitinous biomass exhibited some inhibitory effects on bacterial growth and show potential for use in the medical industry.
- Full Text:
- Date Issued: 2019
- Authors: Oree, Glynis
- Date: 2019
- Subjects: Chitin -- Biotechnology , Enzymes -- Biotechnology , Hydrolysis , Chitooligomers -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67887 , vital:29165
- Description: There are many diseases and illnesses in the world that require new drug treatments and chitin has been shown to produce chitooligomeric derivatives which exhibit promising antimicrobial and immune-enhancing properties. However, the rate-limiting step is associated with the high recalcitrance of chitinous substrates, and low hydrolytic activities of chitinolytic enzymes, resulting in low product release. To improve and create a more sustainable and economical process, enhancing chitin hydrolysis through various treatment procedures is essential for obtaining high enzyme hydrolysis rates, resulting in a higher yield of chitooligomers (CHOS). In literature, pre-treatment of insoluble biomass is generally associated with an increase in accessibility of the carbohydrate to hydrolytic enzymes, thus generating more products. The first part of this study investigated the effect of alkali- (NaOH) and acid pre-treatments (HCl and phosphoric acid) on chitin biomass, and chemical and morphological modifications were assessed by the employment of scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Energy-Dispersive X-ray spectrometery (EDX) and x-ray diffraction (XRD). Data obtained confirmed that pre-treated substrates were more chemically and morphologically modified. These results confirmed the fact that pre-treatment of chitin disrupts the structure of the biomass, rendering the polymer more accessible for enzymatic hydrolysis. The commercial chitinases from Bacillus cereus and Streptomyces griseus (CHB and CHS) are costly. Bio-prospecting for other chitin-degrading enzymes from alternate sources such as Oidiodendron maius, or the recombinant expression of CHOS, was a more economically feasible avenue. The chit1 gene from Thermomyces lanuginosus, expressed in Pichia pastoris, produced a large range CHOS with a degree of polymerisation (DP) ranging from 1 to above 6. TLC analysis showed that O. maius exhibited chitin-degrading properties by producing CHOS with a DP length of 1 to 3. These two sources were therefore successful in producing chitin-degrading enzymes. The physico-chemical properties of commercial (CHB and CHS) and expressed (Chit1) chitinolytic enzymes were investigated, to determine under which biochemical conditions and on which type of biomass they can function on optimally, for the production of value-added products such as CHOS. Substrate affinity assays were conducted on the un-treated and pre-treated biomass. TLC revealed that chitosan hydrolysis by the commercial chitinases produced the largest range of CHOS with a DP length ranging from 1 to 6. A range of temperatures (35-90oC) were investigated and CHB, CHS and Chit1 displayed optimum activities at 50, 40 and 45 oC, respectively. Thermostability studies that were conducted at 37 and 50oC revealed that CHB and CHS were most stable at 37oC. Chit1 showed great thermostablity at both temperatures, rendering this enzyme suitable for industrial processes at high temperatures. pH optima studies demonstrated that the pH optima for CHB, CHS and Chit1 was at a pH of 5.0, with specific activities of 33.459, 46.2 and 5.776 μmol/h/mg, respectively. The chain cleaving patterns of the commercial enzymes were determined and exo-chitinase activity was exhibited, due to the production of CHOS that were predominantly of a DP length of 2. Enzyme binary synergy studies were conducted with commercial chitinases (CHB and CHS) on colloidal chitin. Studies illustrated that the simultaneous combination of CHB 75%: CHS 25% produced the highest specific activity (3.526 μmol/h/mg), with no synergy. TLC analysis of this enzyme combination over time revealed that predominantly chitobiose was produced. This suggested that the substrate crystallinity and morphology played an important role in the way the enzymes cleaved the carbohydrate. Since CHOS have shown great promise for their antimicrobial properties, the CHOS generated from the chitinous substrates were tested for antimicrobial properties on Bacillus subtilis, Escherichia coli, Klebsiella and Staphlococcus aureus. This study revealed that certain CHOS produced have inhibitory effects on certain bacteria and could potentially be used in the pharamceutical or medical industries. In conclusion, this study revealed that chitinases can be produced and found in alternate sources and be used for the hydrolysis of chitinous biomass in a more sustainabe and economically viable manner. The chitinases investigated (CHB, CHS and Chit1) exhibited different cleaving patterns of the chitinous substrates due to the chemical and morphological properties of the biomass. CHOS produced from chitinous biomass exhibited some inhibitory effects on bacterial growth and show potential for use in the medical industry.
- Full Text:
- Date Issued: 2019
An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
A lignocellulolytic enzyme system for fruit waste degradation : commercial enzyme mixture synergy and bioreactor design
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
Lignocellulosic waste degradation using enzyme synergy with commercially available enzymes and Clostridium cellulovorans XylanaseA and MannanaseA
- Authors: Morrison, David Graham
- Date: 2014
- Subjects: Lignocellulose -- Biodegradation , Enzymes -- Biotechnology , Agricultural wastes as fuel , Polysaccharides -- Biotechnology , Sugar -- Inversion , Clostridium , Xylanases , Monomers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4119 , http://hdl.handle.net/10962/d1013292
- Description: The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.
- Full Text:
- Date Issued: 2014
- Authors: Morrison, David Graham
- Date: 2014
- Subjects: Lignocellulose -- Biodegradation , Enzymes -- Biotechnology , Agricultural wastes as fuel , Polysaccharides -- Biotechnology , Sugar -- Inversion , Clostridium , Xylanases , Monomers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4119 , http://hdl.handle.net/10962/d1013292
- Description: The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.
- Full Text:
- Date Issued: 2014
Bioethanol production from waste paper through fungal biotechnology
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
- Date Issued: 2010
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
- Date Issued: 2010
Cleaning of fouled membranes using enzymes from a sulphidogenic bioreactor
- Authors: Melamane, Xolisa L
- Date: 2004
- Subjects: Membrane filters , Membrane filters -- Fouling , Enzymes -- Biotechnology , Enzymes -- Purification , Water -- Purification -- Membrane filtration , Ultrafiltration
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4133 , http://hdl.handle.net/10962/d1015764
- Description: Maintenance of membrane performance requires inevitable cleaning or defouling of fouled membranes. Membrane cleaning using enzymes such as proteases, lipases, α-glucosidases from a sulphidogenic bioreactor was investigated. At first, dilute and concentrated enzyme extract were prepared form the sulphidogenic pellet. Enzyme assays on 0.5 % azocaisen, 1 % triacetin and 1 mg/ml ρ-nitrophenyl-α-D-glucopyranoside were performed using the concentrated enzyme extract (0 – 200 mg/ml). For membrane fouling, an abattoir effluent was obtained from Ostritech Pty (Ltd), Grahamstown, South Africa. The effluent was characterised for presence of potential foulants such as lipids, proteins, amino acids and carbohydrates. Static fouling of polysulphone membranes (0.22 μm, 47 mm) was then performed using the abattoir effluent. Cleaning of the fouled membranes was also performed using at first the dilute and then the concentrated form (200 mg/ml) of enzyme extracts. Qualitative and quantitative biochemical analysis for proteins, lipids and carbohydrates was performed to ascertain the presence of foulants on polysulphone membranes and their removal by dilute or concentrated enzyme extracts. The ability of dilute enzyme extracts to remove proteins lipids, and carbohydrates fouling capillary UF membrane module; their ability to restore permeate fluxes and transmembrane pressure after cleaning/defouling was also investigated. Permeate volumes from this UF membrane module were analysed for protein, amino acids, lipids, and carbohydrates concentrations after fouling and defouling. Fouling was further characterized by standard blocking, cake filtration and pore blocking models using stirred UF cell and polyethersulphone membranes with MWCO of 30 000, 100 000 and 300 000. After characterization of fouling, polyethersulphone membranes with MWCO of 30 000 and 300 000 were defouled using the concentrated enzyme extract (100 mg ml). Enzyme activities at 200 mg/ml of enzyme concentration were 8.071 IU, 86.71 IU and 789.02 IU for proteases, lipases and α-glucosidases. The abattoir effluent contained 553 μg/ml of lipid, 301 μg/ml of protein, 141 μg/ml of total carbohydrate, and 0.63 μg/ml of total reducing sugars. Proteins, lipids and carbohydrates fouling polysulphone membranes after a day were removed by 23.4 %, when a dilute enzyme was used. A concentrated enzyme extract of 200 mg/ml was able to remove proteins, lipids and carbohydrates up to 5 days of fouling by 100 %, 82 %, 71 %, 68 % and 76 % respectively. Defouling of dynamically fouled capillary ultrafiltration membranes using sulphidogenic proteases was successful at pH 10, 37°C, within 1 hour. Sulphidogenic proteases activity was 2.1 U/ml and flux Recovery (FR %) was 64. Characterization of fouling revealed that proteins and lipids were major foulants while low concentration of carbohydrates fouled polyethersulphone membranes. Fouling followed standard blocking for 10 minutes in all the membranes; afterwards fouling adopted cake filtration model for membranes with 30 000 MWCO and pore blocking model for membranes with 300 000 MWCO. A concentration of 100 mg/ml of enzyme extract was able to remove fouling from membranes with MWCO of 30 000. Defouling membranes that followed pore blocking model i.e. 300 000 MWCO was not successful due to a mass transfer problem. From the results of defouling of 30 000 and 300 000 MWCO it was concluded that defouling of cake layer fouling (30 000 MWCO) was successful while defouling of pore blocking fouling was unsuccessful due to a mass transfer problem. The ratio of enzymes present in the enzyme extract when calculated based on enzymatic activity for proteases, lipases and α-glucosidases was 1.1 %, 11 % and 87.9 %. It was hypothesized that apart from proteases, lipases, α and β-glucosidases; phosphatases, sulphatases, amonipeptidases etc. from a sulphidogenic bioreactor clean or defoul cake layer fouling by organic foulants and pore blocking fouling provided the mass transfer problem is solved. However, concentration of enzymes from a sulphidogenic bioreactor has not been optimized yet. Other methods of concentrating the enzyme extract can be investigated for example use of organic solvents.
- Full Text:
- Date Issued: 2004
- Authors: Melamane, Xolisa L
- Date: 2004
- Subjects: Membrane filters , Membrane filters -- Fouling , Enzymes -- Biotechnology , Enzymes -- Purification , Water -- Purification -- Membrane filtration , Ultrafiltration
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4133 , http://hdl.handle.net/10962/d1015764
- Description: Maintenance of membrane performance requires inevitable cleaning or defouling of fouled membranes. Membrane cleaning using enzymes such as proteases, lipases, α-glucosidases from a sulphidogenic bioreactor was investigated. At first, dilute and concentrated enzyme extract were prepared form the sulphidogenic pellet. Enzyme assays on 0.5 % azocaisen, 1 % triacetin and 1 mg/ml ρ-nitrophenyl-α-D-glucopyranoside were performed using the concentrated enzyme extract (0 – 200 mg/ml). For membrane fouling, an abattoir effluent was obtained from Ostritech Pty (Ltd), Grahamstown, South Africa. The effluent was characterised for presence of potential foulants such as lipids, proteins, amino acids and carbohydrates. Static fouling of polysulphone membranes (0.22 μm, 47 mm) was then performed using the abattoir effluent. Cleaning of the fouled membranes was also performed using at first the dilute and then the concentrated form (200 mg/ml) of enzyme extracts. Qualitative and quantitative biochemical analysis for proteins, lipids and carbohydrates was performed to ascertain the presence of foulants on polysulphone membranes and their removal by dilute or concentrated enzyme extracts. The ability of dilute enzyme extracts to remove proteins lipids, and carbohydrates fouling capillary UF membrane module; their ability to restore permeate fluxes and transmembrane pressure after cleaning/defouling was also investigated. Permeate volumes from this UF membrane module were analysed for protein, amino acids, lipids, and carbohydrates concentrations after fouling and defouling. Fouling was further characterized by standard blocking, cake filtration and pore blocking models using stirred UF cell and polyethersulphone membranes with MWCO of 30 000, 100 000 and 300 000. After characterization of fouling, polyethersulphone membranes with MWCO of 30 000 and 300 000 were defouled using the concentrated enzyme extract (100 mg ml). Enzyme activities at 200 mg/ml of enzyme concentration were 8.071 IU, 86.71 IU and 789.02 IU for proteases, lipases and α-glucosidases. The abattoir effluent contained 553 μg/ml of lipid, 301 μg/ml of protein, 141 μg/ml of total carbohydrate, and 0.63 μg/ml of total reducing sugars. Proteins, lipids and carbohydrates fouling polysulphone membranes after a day were removed by 23.4 %, when a dilute enzyme was used. A concentrated enzyme extract of 200 mg/ml was able to remove proteins, lipids and carbohydrates up to 5 days of fouling by 100 %, 82 %, 71 %, 68 % and 76 % respectively. Defouling of dynamically fouled capillary ultrafiltration membranes using sulphidogenic proteases was successful at pH 10, 37°C, within 1 hour. Sulphidogenic proteases activity was 2.1 U/ml and flux Recovery (FR %) was 64. Characterization of fouling revealed that proteins and lipids were major foulants while low concentration of carbohydrates fouled polyethersulphone membranes. Fouling followed standard blocking for 10 minutes in all the membranes; afterwards fouling adopted cake filtration model for membranes with 30 000 MWCO and pore blocking model for membranes with 300 000 MWCO. A concentration of 100 mg/ml of enzyme extract was able to remove fouling from membranes with MWCO of 30 000. Defouling membranes that followed pore blocking model i.e. 300 000 MWCO was not successful due to a mass transfer problem. From the results of defouling of 30 000 and 300 000 MWCO it was concluded that defouling of cake layer fouling (30 000 MWCO) was successful while defouling of pore blocking fouling was unsuccessful due to a mass transfer problem. The ratio of enzymes present in the enzyme extract when calculated based on enzymatic activity for proteases, lipases and α-glucosidases was 1.1 %, 11 % and 87.9 %. It was hypothesized that apart from proteases, lipases, α and β-glucosidases; phosphatases, sulphatases, amonipeptidases etc. from a sulphidogenic bioreactor clean or defoul cake layer fouling by organic foulants and pore blocking fouling provided the mass transfer problem is solved. However, concentration of enzymes from a sulphidogenic bioreactor has not been optimized yet. Other methods of concentrating the enzyme extract can be investigated for example use of organic solvents.
- Full Text:
- Date Issued: 2004
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