title: Condensation corrosion alters the oxygen and carbon isotope ratios of speleothem and limestone surfaces

authors: White, Jackson H.

abstract: Condensation corrosion is a natural process which enhances the chemical weathering of limestone cave chambers and speleothems. We evaluated the use of carbonate tablets for detecting condensation corrosion in Glowworm Cave, New Zealand, using local limestone and speleothem as experimental substrates (herein tablets). Evidence for condensation corrosion was assessed via three methods: gravimetric (mass wasting), microscopic (surface pitting, recrystallization) and isotopic (δ¹³C and δ¹⁸O changes). Our results show little evidence of tablet mass loss throughout a 6-month deployment period. However, SEM imaging and isotope analysis (δ¹³C and δ¹⁸O) of the upper ∼50 μm layer of the tablets, suggest that condensation corrosion operates in the cave, especially in sectors affected by large diurnal microclimate variations.

Most notably, condensation water altered the tablet surface δ¹³C and δ¹⁸O values. Small, positive shifts in surface δ¹³C and δ¹⁸O values are considered to reflect pure dissolution (where dissolution favours the removal of lighter isotopologues). In contrast, tablets that exhibited large positive shifts in δ¹³C in tandem with large negative shifts in δ¹⁸O values, are interpreted as showing calcite recrystallization and the inheritance of higher DIC δ¹³C values (¹³C fractionation by CO₂ degassing), lighter water δ¹⁸O values and/or kinetic fractionation of δ¹⁸O. This study therefore demonstrates that stable isotopes could be applied to detect paleoclimatic episodes of condensation corrosion in speleothems.

text: Condensation corrosion alters the oxygen and carbon isotope ratios of speleothem and limestone surfaces Condensation corrosion is a natural process which enhances the chemical weathering of limestone cave chambers and speleothems. We evaluated the use of carbonate tablets for detecting condensation corrosion in Glowworm Cave, New Zealand, using local limestone and speleothem as experimental substrates (herein tablets). Evidence for condensation corrosion was assessed via three methods: gravimetric (mass wasting), microscopic (surface pitting, recrystallization) and isotopic (δ¹³C and δ¹⁸O changes). Our results show little evidence of tablet mass loss throughout a 6-month deployment period. However, SEM imaging and isotope analysis (δ¹³C and δ¹⁸O) of the upper ∼50 μm layer of the tablets, suggest that condensation corrosion operates in the cave, especially in sectors affected by large diurnal microclimate variations.

Most notably, condensation water altered the tablet surface δ¹³C and δ¹⁸O values. Small, positive shifts in surface δ¹³C and δ¹⁸O values are considered to reflect pure dissolution (where dissolution favours the removal of lighter isotopologues). In contrast, tablets that exhibited large positive shifts in δ¹³C in tandem with large negative shifts in δ¹⁸O values, are interpreted as showing calcite recrystallization and the inheritance of higher DIC δ¹³C values (¹³C fractionation by CO₂ degassing), lighter water δ¹⁸O values and/or kinetic fractionation of δ¹⁸O. This study therefore demonstrates that stable isotopes could be applied to detect paleoclimatic episodes of condensation corrosion in speleothems.

year: 2021

title: Caldolase: an alkaline serine protease from Thermus strain TOK₃

authors: Saravani, G.A.

abstract: The object of this investigation was the isolation of extreme thermophiles producing extracellular proteases, and the biochemical characterisation of a stable, chelator-insensitive protease. 
Two plate assay systems were developed for the initial screening of proteases. The first involved the incorporation of various protease inhibitors (particularly chelating agents) in casein agar plates, the second the inclusion of a variety of native and chromogenic proteins in the agar plates. In conjunction, these methods provided a basis for screening extreme thermophiles for particular proteases, and enabled the identification of fourteen new proteases. Included amongst them was an extracellular serine protease from a Thermus strain designated Tok₃, which was selected for further study.
The protease was purified to homogeneity by ammonium-sulphate precipitation followed by ion exchange on DEAE-cellulose and QAE-Sephadex, affinity chromatography on CBZ-phe-TETA-Sepharose-4B, gel filtration chromatography on either Sephadex G-75 or TSK G3000 SW using HPLC system and finally Polyacrylamide gel electrophoresis.
For convenience the protease was assigned a trivial name, and the term Caldolase was chosen. The prefix Caldo- is derived from Latin Caldo (hot) and the suffix ‘ase’ is a general term for enzymes. 
The specific activity of the pure enzyme was estimated to be 38,000 proteolytic units per mg (PU mg ml⁻¹) at 75°C using casein as the substrate. The purified enzyme had a pH optimum of 9.5 with an isoelectric point of 8.9. Caldolase demonstrated greatest stability between pH 7 and 10. The molecular weight of the protease was estimated by exclusion chromatography on Sephadex G-75 and TSK G3000 SW to be 25,000 daltons.
The enzyme was inhibited by serine inhibitors (DFP, PMSF and di-phenyl carbamyl chloride), partially inhibited by heavy metal (CUCl₂), but not inhibited by metal chelators (EDTA, EGTA, 0-phenanthroline, and NEPIS), Cysteine inhibitors (PCMB, iodo-acetamide, and N-ethylmaleimide) or trypsin inhibitors. These results indicate that Caldolase is an alkaline serine protease. 
Neither Ca²⁺ nor Zn²⁺ ions were detected in the highly purified protease. The presence of four disulphide bonds per molecule of the enzyme was indicated with dithionitro-benzoate. No free sulphydryl groups were found. The purified protease contained approximately 10% carbohydrate. The amino acid composition of Caldolase was determined. 
The enzyme exhibited strong substrate inhibition when using casein, azo-casein, and azo-albumin as substrates. No substrate inhibition was observed when low-molecular weight synthetic substrates were used, indicating that substrate inhibition using casein and azo-albumin substrates may be due to steric hindrance rather than binding to the active site of the enzyme.
The Arrhenius plots for both casein and peptide substrates were curved, but without any clearly marked discontinuity. It is concluded that the effect of temperature on the enzyme conformation is continuous rather than occurring at a particular temperature. No significant differences were observed in Kₘ values at various temperatures between 45° and 85°C.
Caldolase hydrolysed several protein substrates, including casein, albumin, ovalbumin, haemoglobin, collagen, fibrin and elastin, and a number of synthetic chromogenic peptides. It also possessed esterase activity. The enzyme was not able to hydrolyse peptides possessing fewer than four groups (amino acid residues and terminal blocking group). Caldolase did not hydrolyse Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-phe-Arg). In contrast enzyme hydrolysis of insulin B chain resulted in a very complex pattern, suggesting a low degree of specificity. 
The enzyme was very thermostable. Half-life values were: 100°C, 5 min; 90°C, 45 min; and 80°C, 840 min. Caldolase was stable in denaturing agents (GuHCl, urea) at 22°C, but not at 85°C. Exposure of the enzyme to various organic solvents caused no significant loss of catalytic activity.
Ionic strength had a marked effect on enzyme stability. The combination of low salt concentration (below 0.3M NaCl) and low temperatures (under 75°C) results in reversible enzyme denaturation. However, at high temperatures (above 80°C) this phenomenon is rapidly followed by autolysis by the remaining active enzyme.

text: Caldolase: an alkaline serine protease from Thermus strain TOK₃ The object of this investigation was the isolation of extreme thermophiles producing extracellular proteases, and the biochemical characterisation of a stable, chelator-insensitive protease. 
Two plate assay systems were developed for the initial screening of proteases. The first involved the incorporation of various protease inhibitors (particularly chelating agents) in casein agar plates, the second the inclusion of a variety of native and chromogenic proteins in the agar plates. In conjunction, these methods provided a basis for screening extreme thermophiles for particular proteases, and enabled the identification of fourteen new proteases. Included amongst them was an extracellular serine protease from a Thermus strain designated Tok₃, which was selected for further study.
The protease was purified to homogeneity by ammonium-sulphate precipitation followed by ion exchange on DEAE-cellulose and QAE-Sephadex, affinity chromatography on CBZ-phe-TETA-Sepharose-4B, gel filtration chromatography on either Sephadex G-75 or TSK G3000 SW using HPLC system and finally Polyacrylamide gel electrophoresis.
For convenience the protease was assigned a trivial name, and the term Caldolase was chosen. The prefix Caldo- is derived from Latin Caldo (hot) and the suffix ‘ase’ is a general term for enzymes. 
The specific activity of the pure enzyme was estimated to be 38,000 proteolytic units per mg (PU mg ml⁻¹) at 75°C using casein as the substrate. The purified enzyme had a pH optimum of 9.5 with an isoelectric point of 8.9. Caldolase demonstrated greatest stability between pH 7 and 10. The molecular weight of the protease was estimated by exclusion chromatography on Sephadex G-75 and TSK G3000 SW to be 25,000 daltons.
The enzyme was inhibited by serine inhibitors (DFP, PMSF and di-phenyl carbamyl chloride), partially inhibited by heavy metal (CUCl₂), but not inhibited by metal chelators (EDTA, EGTA, 0-phenanthroline, and NEPIS), Cysteine inhibitors (PCMB, iodo-acetamide, and N-ethylmaleimide) or trypsin inhibitors. These results indicate that Caldolase is an alkaline serine protease. 
Neither Ca²⁺ nor Zn²⁺ ions were detected in the highly purified protease. The presence of four disulphide bonds per molecule of the enzyme was indicated with dithionitro-benzoate. No free sulphydryl groups were found. The purified protease contained approximately 10% carbohydrate. The amino acid composition of Caldolase was determined. 
The enzyme exhibited strong substrate inhibition when using casein, azo-casein, and azo-albumin as substrates. No substrate inhibition was observed when low-molecular weight synthetic substrates were used, indicating that substrate inhibition using casein and azo-albumin substrates may be due to steric hindrance rather than binding to the active site of the enzyme.
The Arrhenius plots for both casein and peptide substrates were curved, but without any clearly marked discontinuity. It is concluded that the effect of temperature on the enzyme conformation is continuous rather than occurring at a particular temperature. No significant differences were observed in Kₘ values at various temperatures between 45° and 85°C.
Caldolase hydrolysed several protein substrates, including casein, albumin, ovalbumin, haemoglobin, collagen, fibrin and elastin, and a number of synthetic chromogenic peptides. It also possessed esterase activity. The enzyme was not able to hydrolyse peptides possessing fewer than four groups (amino acid residues and terminal blocking group). Caldolase did not hydrolyse Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-phe-Arg). In contrast enzyme hydrolysis of insulin B chain resulted in a very complex pattern, suggesting a low degree of specificity. 
The enzyme was very thermostable. Half-life values were: 100°C, 5 min; 90°C, 45 min; and 80°C, 840 min. Caldolase was stable in denaturing agents (GuHCl, urea) at 22°C, but not at 85°C. Exposure of the enzyme to various organic solvents caused no significant loss of catalytic activity.
Ionic strength had a marked effect on enzyme stability. The combination of low salt concentration (below 0.3M NaCl) and low temperatures (under 75°C) results in reversible enzyme denaturation. However, at high temperatures (above 80°C) this phenomenon is rapidly followed by autolysis by the remaining active enzyme.

year: 1985

title: Boat electrofishing survey of fish abundance in the Ohau Channel, Rotorua, in 2015

authors: Hicks, Brendan J.

abstract: The aim of the survey was to provide on-going monitoring of the fish communities and abundance by boat electrofishing in the Ohau Channel, especially fish species that are taonga to Maori (eels, goldfish, and koura). In the current study, we present the findings from the ninth year of sampling (2015) and a summary of previous surveys.
We used the University of Waikato's 4.5 m-long, aluminium-hulled electrofishing boat to catch a total of total of 1,198 fish and koura (18.9 kg) at 13 sites on 2 December 2015, which comprised 2,671 lineal m and 10,684 m² in area. Koura (freshwater crayfish) and 6 fish species were present, with common bully the most abundant species (up to 45.5 fish 100 m⁻² at the site 6, edge habitat). Goldfish (up to 4.85 100 m⁻²) was the next most abundant species, with most goldfish at sites 7 and 12-13 in and around the side channel. Rainbow trout densities were up to 0.28 fish 100 m⁻². Mean bully density (11.41 fish 100 m⁻²) was much higher than for smelt (0.29 fish 100 m⁻²). Koura had a patchy distribution; only 3 individuals were caught at one site.
Comparing catches over the 9 years of sampling, the mean abundance of common bullies in 2015 was consistent with densities in most post-wall years (after 2007), but lower than in 2007 before wall closure (ANOVA P = 0.001). The cause of fluctuating bully abundance is not known, and was not accounted for by changes in water clarity expressed as black disc distance (BDD), water temperature, or water conductivity. Poor water clarity can reduce the efficiency of electrofishing, but high BDD did not correspond with high common bully densities. In 2015, smelt abundance had recovered somewhat from the low catch in 2014.
Goldfish biomass increased initially (2009-2010) because of targeted fishing in the excavated side branch (site 11), which has dense macrophytes and offers good habitat for goldfish. The continued rise in density from 2012 on, however, suggests a real increase in goldfish numbers. In 2012 and 2013 shortfin eels were caught, but no eels were caught in 2014 and only a single longfin eel was caught in 2015.
Analysis of fish densities before and after wall closure is hampered by the single data point before closure. However, we now have 9 years of post-wall data, and comparison of means suggest that the number of bullies has been lower since 2007. An obvious cause could be interruption of bully migration into the Ohau Channel from Lake Rotoiti by the wall. This suggests that the bully population in the Ohau Channel before wall construction and closure was a mixture of fish from lakes Rotorua and Rotoiti, and that recruitment from Rotoiti is now restricted. This hypothesis is testable with otolith microchemistry.

text: Boat electrofishing survey of fish abundance in the Ohau Channel, Rotorua, in 2015 The aim of the survey was to provide on-going monitoring of the fish communities and abundance by boat electrofishing in the Ohau Channel, especially fish species that are taonga to Maori (eels, goldfish, and koura). In the current study, we present the findings from the ninth year of sampling (2015) and a summary of previous surveys.
We used the University of Waikato's 4.5 m-long, aluminium-hulled electrofishing boat to catch a total of total of 1,198 fish and koura (18.9 kg) at 13 sites on 2 December 2015, which comprised 2,671 lineal m and 10,684 m² in area. Koura (freshwater crayfish) and 6 fish species were present, with common bully the most abundant species (up to 45.5 fish 100 m⁻² at the site 6, edge habitat). Goldfish (up to 4.85 100 m⁻²) was the next most abundant species, with most goldfish at sites 7 and 12-13 in and around the side channel. Rainbow trout densities were up to 0.28 fish 100 m⁻². Mean bully density (11.41 fish 100 m⁻²) was much higher than for smelt (0.29 fish 100 m⁻²). Koura had a patchy distribution; only 3 individuals were caught at one site.
Comparing catches over the 9 years of sampling, the mean abundance of common bullies in 2015 was consistent with densities in most post-wall years (after 2007), but lower than in 2007 before wall closure (ANOVA P = 0.001). The cause of fluctuating bully abundance is not known, and was not accounted for by changes in water clarity expressed as black disc distance (BDD), water temperature, or water conductivity. Poor water clarity can reduce the efficiency of electrofishing, but high BDD did not correspond with high common bully densities. In 2015, smelt abundance had recovered somewhat from the low catch in 2014.
Goldfish biomass increased initially (2009-2010) because of targeted fishing in the excavated side branch (site 11), which has dense macrophytes and offers good habitat for goldfish. The continued rise in density from 2012 on, however, suggests a real increase in goldfish numbers. In 2012 and 2013 shortfin eels were caught, but no eels were caught in 2014 and only a single longfin eel was caught in 2015.
Analysis of fish densities before and after wall closure is hampered by the single data point before closure. However, we now have 9 years of post-wall data, and comparison of means suggest that the number of bullies has been lower since 2007. An obvious cause could be interruption of bully migration into the Ohau Channel from Lake Rotoiti by the wall. This suggests that the bully population in the Ohau Channel before wall construction and closure was a mixture of fish from lakes Rotorua and Rotoiti, and that recruitment from Rotoiti is now restricted. This hypothesis is testable with otolith microchemistry.

year: 2016