Plant-fungal mutualism as a strategy for the bioremediation of hydrocarbon polluted soils
- Authors: Keshinro, Olajide Muritala
- Date: 2021-10-29
- Subjects: Mutualism (Biology) , Plant-fungus relationships , Bioremediation , Mucilage , Plant exudates , Extracellular polymeric substances , Laccase , Peroxidase , Phytoremediation , Ligninolytic enzymes
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/190918 , vital:45041 , 10.21504/10962/190918
- Description: Inasmuch as coal remains the linchpin for the generation of electricity and liquid petroleum products in South Africa, hydrocarbon waste and coal discard will continue to pose a threat to the environment. Therefore, the onus is on the associated industries to develop and implement efficient and sustainable strategies to mitigate the negative impacts of energy generating activities on the environment. Most conventional efforts in this regard, although successful for soil repair and the initiation of vegetation, have been deemed unsustainable. In an effort to find a sustainable remediation strategy a novel technology termed “FungCoal” was conceptualized and patented as a strategy for the rehabilitation of open cast coal mines, carbonaceous-rich spoils and coal wastes. This biotechnology, which exploits plant-fungal mutualism to achieve effective biodegradation of coal on discard dumps and the breakdown of the carbonaceous component in spoils, promotes revegetation to facilitate rehabilitation of mining-disturbed land. However, one limiting factor of the FungCoal bioprocess is that it requires oxidized weathered coal, a highly complex and variable resource for use as a co-substrate, for growth and proliferation of the coal degrading microorganisms. To fully exploit the potential of plant-fungal mutualism and its interaction for use in the remediation of coal contaminated soils, this study investigated the proposed relationship between plant roots, root exudate and the coal degrading fungus “Aspergillus sp.” (previously Neosartorya fischeri) strain 84 in more detail, in an effort to gain further insight into the mechanisms underpinning plant-fungal mutualism as a strategy for re-vegetation of coal discard dumps and the rehabilitation of hydrocarbon-contaminated soil using the FungCoal approach. A pot-on-beaker (PoB) method was developed for the easy cultivation and collection of extracellular polymeric substance (EPS)-containing exudates from Zea mays L. (maize) and Abelmuschus esculentus (okra). Characterisation of the EPS material from these exudates was carried out using a combination of physicochemical and biochemical methods. The results from analysis of phenolics and indoles showed that exudates contain some form of indoles and phenolic compounds, although in little proportions, which may fulfil a signalling function, responsible for attracting soil microorganisms into the rhizosphere. Spectroscopic analysis of the exudates using FT-IR revealed vibrations corresponding to functional groups of alkanes, alkenes, alkynes, and carboxylic acids. These compounds likely provide an easily accessible source of carbon to soil microorganisms and are also a better alternative to the poly-aromatics which are an inherent component locked-up in the supposed recalcitrant coal material. The results from biochemical analyses also revealed the presence of carbohydrate, proteins, lipids, and low amounts of α-amino-nitrogen in the EPS of maize and okra. These components of EPS are all essential for the stimulation of enzymatic activities in soil microorganisms and, which may in turn aid biodegradation. The action of the root EPS from maize was further tested on three coal-degrading fungal isolates identified as Aspergillus strain ECCN 84, Aspergillus strain ECCN 225 and Penicillium strain ECCN 243 for manganese peroxidase (MnP) and laccase (LAC) activities. The results revealed that the Aspergillus species, strains ECCN 84 and ECCN 225, showed with or without EPS, observable black halos surrounding each of the colonies after 7d incubation indicative of positive MnP activity, while no activity was observed for the Penicillium sp. strain ECCN 243. Analysis for LAC revealed little or no activity in any of the coal degrading fungi following addition of pulverized coal to the growth medium. Interestingly, the addition of EPS-containing exudate to the coal-containing medium resulted in increased LAC activity for all fungal isolates. This finding affirmed the positive contribution of EPS to extracellular LAC activity, purported as an important enzyme in the coal biodegradation process. Finally, the impact of plant-derived exudate on the colonisation and biodegradation of coal was investigated in situ using rhizoboxes, to simulate a coal environment, and was carried out for 16 weeks. Microscopic examination of coal samples after termination of the experiment showed fungal proliferation and attachment to coal particles. All of the rhizoboxes that contained plants had higher medium pH and EC, and the concentration of phenolics, indoles and humic acids was greater than that of control treatments. These observations indicated better rhizosphere colonisation, substrate biodegradation and humification. Therefore, root exudate appears to play a significant role in coordination of soil microorganisms within the rhizosphere and likely serves both as a scaffold for rhizospheric interactions by providing microorganisms with accessible carbon and as a likely ‘trigger’ for induction of coal-degrading enzymes such as fungal LAC for mobilisation of recalcitrant carbon. This study has shown that EPS exuded from roots of Zea mays together with coal degrading fungus Aspergillus strain ECCN 84 can alkalinise the coal substrate and facilitate introduction of oxygen, possibly as a result of increased laccase activity, and increase availability of nutrients (as indicated by higher EC) in a coal-polluted rhizosphere, to provide plants and their associated mycorrhizae and presumably other beneficial microorganisms a more mesic environment for sustained phytoremediation with enhanced rehabilitation potential. In conclusion, this study confirms the positive role of root exudate in mediating a mutualistic rehabilitation strategy involving plants and fungi such as the FungCoal bioprocess. , Thesis (PhD) -- Faculty of Science, Institute for Environmental Biotechnology, 2021
- Full Text:
- Date Issued: 2021-10-29
Waste keratinous biomass valorization and characterization of keratinases produced by exiguobacteria species
- Authors: Dlume, Tutuka
- Date: 2021-02
- Subjects: Factory and trade waste -- Biodegradation , Bioremediation
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20695 , vital:46438
- Description: Keratinous wastes are emanating in a million tons, as by-products, from various agro-industrial processing plants. Consequently, they create a serious solid waste problem in the environment due to poor handling. Microbial keratinases are proteolytic enzymes that effectively participate in keratin-rich biomass hydrolyses such as feathers, nail, hair, hooves, and horns. Therefore, proper management of these wastes via recycling into useful products is ecologically imperative. Biodegradation of keratin-rich biomass has been identified as an economical and environmentally friendly way of transforming these recalcitrant agro wastes into useful products, hence the motivation for this study. Feather degrading bacterial strains previously isolated from a municipal dumpsite and coded as SSB-02 and SSB-03 was identified through 16S rDNA sequencing and phylogenetic analysis. The fermentation conditions for keratinase production were optimized. The protein and amino acids constituents of the hydrolyzed chicken feather were analyzed. The biochemical properties of the keratinase produced were determined. Also, the effect of laundry detergents on the stability of the keratinase was studied. The isolates coded as SSB-02 and SSB-03 showed a high percentage of sequence homology with Exguobacterium spp., hence they were identified as Exiguobacterium sp. FBH5 and Exiguobacterium acetylicum FHBD, respectively. Exiguobacterium sp. FBH5 showed the highest extracellular keratinase production of 934.58 ± 27.27 U/mL at 72 h of incubation; in optimized fermentation conditions that included pH (5.0), temperature (30 oC), and chicken feather (0.5percent, w/v). Similarly, E. acetylicum FHBD displayed optimal keratinase production of 1023.64 ± 25.71 U/mL at 120 h of fermentation and improved fermentation conditions that involved pH (3.0), temperature (35 oC) and chicken feathers (0.5-1.5percent; w/v). The amino acid analysis showed that arginine, aspartic acid and glutamic acid were the most abundant amino acids cleaved from the degradation of chicken feathers by Exiguobacterium sp. FBH5 at a respective concentration of 1.16, 1.28 and 1.45 (g/100g sample). Additionally, hydrolysate that emanated from E. indicum FHBD degradation of feather showed high concentrations of arginine, serine, aspartic acid, glutamic acid, and glycine at the respective concentration (g/100g sample) of 1.2, 1.12, 1.34, 1.58 and 1.29. The keratinases were optimally active at pH and temperature of 8.0 and 50 oC, respectively. EDTA and PMSF did not highly repress keratinolytic proteases' activity, and this inhibitory pattern suggests that they may belong to a mixed protease family. Keratinase from E. acetylicum FHBD was highly stable in the presence of SDS, with 99percent residual activity and displayed variable stability in other chemical agents tested. A similar stability pattern was observed with keratinase from Exiguobacterium sp. FBH5, although the enzyme lost about 40percent of its original activity in the presence of SDS. Evaluation of metal ion stability indicated that E. acetylicum FHBD keratinase was remarkably stable in the presence of Fe3+, Mg2+, Cu2+, Zn2+, and Ba2+, with residual activity of 94percent, 88percent, 89percent, 90percent, and 97percent, respectively. Similarly, Exiguobacterium sp. FBH5 keratinase was considerably stable after treatment with Co2+, K+, and Zn2+ as it displayed a residual activity of 85percent, 84percent and 93percent, respectively. The study of the keratinases stability in laundry detergents showed that E. acetylicum FHBD keratinolytic proteases was activated in the presence of Omo, Surf, Sunlight, and Pro wash after 60 min of pre-incubation compared to 30 min, with residual activity of 94 ± 2.94percent, 91 ± 2.53percent, 95 ± 2.89percent and 87 ± 2.89percent respectively. Likewise, Exiguobacterium sp. FBH5 keratinase activity was promoted after 60 min of incubation compared to 30 min, with a residual enzyme activity of 79percent, 84percent, 101percent, 103percent and 105percent and 106percent for Ariel, Surf, Prowash, Freewave, Sky and Evaklin, respectively. Therefore Exiguobacterium spp., demonstrated excellent keratinolytic potentials that could be exploited for sustainable development of bio-innovative products. The study keratinases' properties suggest their industrial and biotechnological application potentials, especially as bio-additive in the formulation of laundry detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
Performance evaluation and cost analysis of subsurface flow constructed wetlands designed for ammonium-nitrogen removal
- Authors: Tebitendwa, Sylvie Muwanga
- Date: 2018
- Subjects: Sewage Purification Nitrogen removal , Constructed wetlands , Bioremediation , Sewage lagoons , Coal mine waste
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61808 , vital:28062
- Description: Subsurface flow constructed wetlands (SSF CWs) is a low-cost, environmentally friendly sanitation technology for on-site treatment of domestic/municipal sewage. However, these systems are apparently unable to produce treated water of a quality suitable for discharge particularly in terms of nitrogen concentration, which has been attributed to design and operation based on biological oxygen demand as the parameter of choice. The aim of this study was to evaluate the performance, support medium, and techno-economics of a vertical- horizontal (V-H) SSF hybrid CW designed and operated using ammonium-nitrogen (NH4+-N) as the major parameter. Two pilot scale V-H SSF hybrid CWs were designed, constructed, and the performance of each monitored over two seasons and under two phases i.e. an initiation phase, and an optimization phase. Laboratory-scale horizontal SSF CWs were used to evaluate the support medium while the techno-economic study was framed to determine the cost effectiveness of V-H SSF hybrid CWs relative to high rate algal oxidation pond (HRAOP) systems to increase capacity of overloaded and/or under-performing waste stabilization pond (WSP) sewage treatment plants. Results revealed that under optimal operating conditions of hydraulic loading rate, hydraulic retention, and influent NH4+-N loading rate, treated water from the V-H SSF hybrid CWs achieved a quality commensurate with current South African standards for discharge into a surface water resource for all parameters except chemical oxygen demand and faecal coliforms. This suggests that NH4+-N is an important design and operational parameter for SSF CWs treating municipal sewage that is characterised as weak in terms of NH4+-N with a requirement of only simple disinfection such as chlorination to eliminate faecal coliforms. Use of discard coal to replace gravel as support medium in horizontal SSF CWs revealed an overall reduction in elemental composition of the discard coal support medium but without compromising water quality. This result strongly supports use of discard coal as an appropriate substrate for SSF CWs to achieve acceptable water quality. Furthermore, simultaneous degradation of discard coal during wastewater treatment demonstrates the versatility of SSF CWs for use in bio-remediation and pollution control. Finally, a technoeconomic assessment of V-H SSF hybrid CWs and a HRAOP series was carried out to determine the suitability of each process to increase capacity by mitigating dysfunctional and/or overloaded WSP sewage treatment plants. Analysis revealed that the quality of treated water from both systems was within the South African General Authorization standards for discharge to a surface water resource. Even so, each technology system presented its own set of limitations including; the inability to satisfactorily remove NH4+-N and chemical oxygen demand (i.e. for V-H SSF hybrid CWs) and total suspended solids and faecal coliforms (i.e. for HRAOPs), and a requirement for substantial land footprint while, HRAOPs required significantly less capital than V-H SSF hybrid CWs for implementation. The latter suggests that HRAOPs could be preferred over V-H SSF hybrid CWs as a technology of choice to increase the capacity of overloaded WSP sewage treatment plants especially where financial resources are limited. Overall, the results of this thesis indicate the potential to use NH4+-N as a design parameter in constructing SSF CWs treating weak strength municipal sewage (i.e. in terms of NH4+-N concentration) and to supplant gravel as the treatment media with industrial waste material like discard coal to achieve wastewater treatment, bio-remediation, and pollution control. The results of this work are discussed in terms of using SSF CWs as a passive and resilient technology for the treatment of domestic sewage in sub-Saharan Africa.
- Full Text:
- Date Issued: 2018
Towards the bioremediation of the hypertrophic Swartkops Solar Salt-works
- Authors: Difford, Mark
- Date: 2008
- Subjects: Salt industry and trade -- South Africa -- Port Elizabeth , Bioremediation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10606 , http://hdl.handle.net/10948/1506 , Salt industry and trade -- South Africa -- Port Elizabeth , Bioremediation
- Description: This thesis presents the results of three studies aimed at improving brine-quality at the Swartkops solar salt-works (Swartkops Sea Salt [Pty] Ltd) on the outskirts of Port Elizabeth, South Africa. This is a highly eutrophic salt-works, the management of which has become increasingly difficult in recent years. The fundamental problem is how best to operate the system at maximum capacity while limiting nutrient inputs from the nutrient-rich microtidal Swartkops Estuary. In the first study, brine-quality at several sites along the axis of the Swartkops Estuary, and the extent to which it is affected by a variety of factors, is compared. Sites were sampled on micro- and macrotidal time scales, and were selected by the management of the salt-works as possible locations for a new pump-house (for extracting brine from the estuary) for their salt-work operations at Swartkops and Missionvale. The study showed that there are incremental benefits to be had from moving the site of extraction downstream from its present position to a site closer to the mouth of the estuary, where the concentration of nutrients usually is lower and where salinity usually is higher. There is little to be gained from moving the site of extraction laterally, to the mouth of the Inlet from which brine currently is extracted, so that brine is extracted directly from the estuary itself. A set of models relating the concentrations of NH+ 4 , NO{u100000}3 , and PO34{u100000} to salinity is proposed. These take into account the influences of site and season and may be used to estimate the concentration of these nutrients from a measurement of salinity. The model for PO34{u100000} shows that it would be more damaging to the salt-works’ operations to pump “low”-salinity brine during the early months of summer than during autumn. Evidence is also presented to show that Wylde Bridge has no influence on nutrient concentrations in the estuary, with tidal flushing generally passing beyond the Wylde-Bridge break-point. The exceptionally heavy flooding of the estuary that occurred in September 2002 may, however, have biased this conclusion, because of its scouring effect. The second study concentrated on monitoring the effect of (1) decreasing pond depth and (2) increasing pond salinity—two readily available management tools—on brine quality at the salt-works. Pond depth throughout the salt-works was decreased by 40 cm, and the salinity of Pond 5, a pond in the middle of the system, was increased to 175 S. Both measures were kept in place for the duration of the study (Nov. 2002–Aug. 2004). The pond-depth experiment did not have the expected result, there being no evidence of the increase in microalgal growth in the water column that was predicted based on previous research. There was, however, a significant increase in benthic chlorophyll-a, and there was a general improvement in the condition of the sedimentary system of the salt-works. There was also a substantial decrease in particulate organic matter in the water column, with clear evidence that the remaining fraction was closely associated with living forms of particulate matter rather than with detritus. The pond-salinity experiment proves that there is a flourishing, and resilient, population of brine shrimp (Artemia salina L.) at the salt-works. Restocking the salina, or stocking it with a different strain of brine shrimp, is therefore not necessary. The results of this study show that the brine shrimp population at the salt-works needs salinities of greater than about 65–70 S to survive. As a living force they almost certainly need a protective salinity that is greater than about 120–140 S, perhaps even as great as 160 S. Brine shrimp thrived in the high salinity milieu of the experimental pond for the duration of the study, but dwindled from three other ponds of the system once their salinities fell to below 90 S, eventually to disappear from them, apparently completely, once salinity fell to below 65 S. The third and final study concentrated on establishing whether the products released by decomposing barley straw could be used in a solar salt-works to control macroalgal blooms without detrimentally affecting the benthic-mat. Previous research has shown that these products are effective inhibitors of macroalgal growth and that they remain effective under saline conditions. The results presented here show that the same products, or products released under similar conditions of decomposition, adversely effect both the structure and the function of the mat. Consequently, their use in a solar salt-works cannot be recommended.
- Full Text:
- Date Issued: 2008
Hydrogenases from sulphate reducing bacteria and their role in the bioremediation of textile effluent
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
The bioaccumulation of platinum (IV) from aqueous solution using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Rashamuse, Konanani Justice
- Date: 2003
- Subjects: Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4003 , http://hdl.handle.net/10962/d1004063 , Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Description: The enzymatic reduction of a high-valence form of metals to a low-valence reduced form has been proposed as a strategy to treat water contaminated with a range of metals and radionuclides. Metal reduction by sulphate reducing bacteria (SRB) is carried out either chemically (involving reduction by hydrogen sulphide) or enzymatically (involving redox enzymes such as the hydrogenases). While reduction of metal ions by hydrogen sulphide is well known, the enzymatic mechanism for metal reduction is poorly understood. The aims of this study were to investigate the role of SRB in facilitating platinum removal, and to investigate the role of a hydrogenase in platinum reduction in vitro. In order to avoid precipitation of platinum as platinum sulphide, a resting (non-growing) mixed SRB culture was used. The maximum initial concentration of platinum (IV), which SRB can effectively remove from solution was shown to be 50 mg.l⁻¹. Electron donor studies showed high platinum (IV) uptake in the presence of hydrogen, suggesting that platinum (IV) uptake from solution by SRB requires careful optimization with respect to the correct electron donor. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis indicated that platinum was being precipitated in the periplasm, a major area of hydrogenase activity in SRB. Purification of the hydrogenase by ammonium sulphate precipitation (65%), Toyopearl-Super Q 650S ion exchange and Sephacry 1 S-100 size exclusion chromatography revealed that the hydrogenase was monomeric with a molecular weight of 58 KDa, when analyzed by 12% SDS-PAGE. The purified hydrogenase showed optimal temperature and pH at 35°C and 7.5 respectively, and a poor thermal stability. In vitro investigation of platinum reduction by purified hydrogenase from mixed SRB culture showed that hydrogenase reduces platinum only in the presence of hydrogen. Major platinum (IV) reduction was observed when hydrogenase was incubated with cytochrome C₃ (physiological electron carrier in vivo) under hydrogen. The same observations were also noted with industrial effluent. Collectively these findings suggest that in vitro platinum reduction is mediated by hydrogenase with a concerted action of cytochrome C₃ required to shuttle the electron from hydrogenase.
- Full Text:
- Date Issued: 2003
Spirulina as a bioremediation agent : interaction with metals and involvement of carbonic anhydrase
- Authors: Payne, Rosemary Anne
- Date: 2000
- Subjects: Spirulina , Bioremediation , Carbonic anhydrase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3909 , http://hdl.handle.net/10962/d1003968 , Spirulina , Bioremediation , Carbonic anhydrase
- Description: Heavy metal contamination from mining and other industrial operations is becoming an increasing problem with regards to the depleting water resources in South Africa. This study involved the investigation of the use of an algal biomass as a possible alternative to the traditional chemical means of removing these metals. When the toxic effects of metals were investigated, Spirulina was found to have a threshold level of about 30 μM for copper, zinc and lead. Copper and zinc appeared to have a direct effect on the photosynthetic pathway, thereby causing a rapid decline in cell growth. Lead on the other hand seemed to affect surface properties and hence took longer to cause deterioration in growth. Although relatively low concentrations of metal may have a toxic effect on the cyanobacterium, Spirulina may have potential as a precipitation agent. The role of Spirulina in the precipitation of heavy metals appears to be through its ability to maintain a high pH in the surrounding medium, possibly through the enzyme carbonic anhydrase. Subsequent studies therefore focused on the assay and isolation of this enzyme. Two different radiotracer assays, in which carbonic anhydrase converts radiolabelled bicarbonate to carbon dioxide, were investigated, but were found to have several problems. Results were insensitive and could not be reproduced. The standard Wilbur-Anderson method subsequently investigated also proved to be insensitive with a tremendous degree of variability. Although not quantitative, SDS-PAGE proved to be the most reliable method of detection, and was therefore used in subsequent procedures. Chlamydomonas reinhardtii was the subject of initial enzyme isolation studies as these procedures are well documented. Although the published protocols proved unsuccessful, affinity chromatography of a membrane stock solution from Chlamydomonas reinhardtii yielded two relatively pure protein bands. These bands were presumed to represent two subunits of carbonic anhydrase, although Western blot analysis would be required to confirm their identity. Purification of carbonic anhydrase from Spirulina, however, proved unsuccessful and results obtained were very inconclusive. Hence, further analysis of Spirulina is required. The possibility of cloning CA from a genomic library was also considered, but suitable primers could not be designed from the aligned sequences.
- Full Text:
- Date Issued: 2000