Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.     

Conversion of L-Cystine and L-Cysteine to Taurine by the Enzyme Systems of Liver Cells

The kinetics of the conversion of the sulfur-containing amino acids L-cystine and L-cysteine to taurin by the enzyme system of cattle liver cells was studied and a mathematical model was developed. It was shown that the conversions of L-cystine and L-cysteine obeyed the Michaelis–Menten equations of serial–sequential conversions with regard to inhibition by the final product and inactivation. The yield of taurin under the optimized conditions of L-cystine and L-cysteine conversion (temperature, 40°C; pH 1.5 and 3.0, respectively; and the addition of enzyme preparations in five equal portions at 2-h intervals) was in the range of 80–85% of the substrate weight.     

Purification and characterization of a novel l-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli

l-2-Amino-Δ²-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu²⁺ and Mn²⁺ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K _m (0.35 mM) and V _max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine.     

Cloning,Expression, and Identification of Genes Involved in the Conversion of DL-2-Amino-Δ2-thiazoline-4-carboxylic Acid to L-Cysteine via S-Carbamyl-L-cysteine Pathway in Pseudomonas sp. TS1138

Two novel genes (tsB, tsC) involved in the conversion of DL-2-amino-Δ²-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine through S-carbamyl-L-cysteine (L-SCC) pathway were cloned from the genomic DNA library of Pseudomonas sp. TS1138. The recombinant proteins of these two genes were expressed in Escherichia coli BL21, and their enzymatic activity assays were performed in vitro. It was found that the tsB gene encoded an L-ATC hydrolase, which catalyzed the conversion of L-ATC to L-SCC, while the tsC gene encoded an L-SCC amidohydrolase, which showed the catalytic ability to convert L-SCC to L-cysteine. These results suggest that tsB and tsC play important roles in the L-SCC pathway and L-cysteine biosynthesis in Pseudomonas sp. TS1138, and that they have potential applications in the industrial production of L-cysteine.     

Cloning, expression, characterization and application of atcA, atcB and atcC from Pseudomonas sp. for the production of L-cysteine

An isolate of a Pseudomonas sp. uses the l-NCC (N-carbamoyl-l-cysteine) pathway to convert dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) to l-cysteine. Genes encoding ATC racemase (AtcA), l-ATC hydrolase (AtcB) and l-NCC amidohydrolase (AtcC), involved in this pathway, were cloned from the Pseudomonas sp. and expressed in Escherichia coli BL21 via pET-28a(+). The resulting enzymes were purified, their functions identified, and their biochemical properties are described. In vitro catalysis experiments, using these enzymes, revealed that the bioconversion rate of l-cysteine from dl-ATC in the presence of AtcA was more efficient than in the absence of AtcA. This is the first report describing simultaneous cloning and expression of atcA, atcB and atcC and characterization of their enzymes for l-cysteine production from dl-ATC via the l-NCC pathway, enabling the complete l-NCC pathway to be elucidated.     

Effect of oxygen transfer rate on the composition of the pectolytic enzyme complex of Aspergillus niger

Optimal agitation and aeration conditions [assuring O₂ transfer rates (OTR) from 12 to 179 mmol L^?1 h^?1] were determined for pectin lyase (PL) synthesis of an Aspergillus niger strain. Components of the pectolytic enzyme complex were also investigated in order to determine whether their O₂ demand is identical with or different from that of pectin lyase. Should the latter be the case, a possibility would be given to produce enzyme complexes of different agitation and aeration conditions. According to our results, mycelium yield of Aspergillus niger attained a maximum at an OTR of 100 mmol L^?1 h^?1. The yields of the various pectolytic enzymes reached maxima at different OTRs. Pectin lyase production was the highest (0.555 µmol min^?1 mL^?1) at an OTR of 60 mmol L^?1 h^?1. Endopolygalacturonase (PG) production showed a maximum at the OTR of 49 mmol L^?1 h^?1 with a second peak at 100?135 mmol O₂ L^?1 h^?1. Pectin esterase (PE) synthesis showed a maximum at on OTR of 12?14 mmol L^?1 h^?1, while both apple juice clarifying and macerating activities gave two maxima at 14 and 60 mmol L^?1 h^?1 due to the optima of PE and endo-PG. Macerating activity showed a high value at OTR optimal for PL production as well.     

BspA (CyuC) in Lactobacillus fermentum BR11 Is a Highly Expressed High-Affinity L-Cystine-Binding Protein

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 M. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1–2 × 10⁵ BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.     

Simultaneous Removal of Mn(II) and Nitrate by the Manganese-Oxidizing Bacterium Acinetobacter sp. SZ28 in Anaerobic Conditions

A novel bacterium, strain SZ28, identified as Acinetobacter sp., showed anaerobic denitrification ability using Mn(II) as the electron donor. Nitrate-nitrogen concentration decreased from nearly 16.52–mg L^?1 to 4.4–mg L^?1, without accumulation of nitrite as an intermediate, with a maximum of 0.063–mg NO₃^?-N L^?1 h^?1, reaching a peak of 0.085–mg NO₃^?-N L^?1 h^?1 in sodium acetate. The nitrate removal rate reached 0.067–mg NO₃^?-N L^?1 h^?1, 0.059–mg NO₃^?-N L^?1 h^?1, and 0.078 mg NO₃^?-N L^?1 h^?1 using Mn(II), S(II), and Fe(II) as electron donors, respectively. The optimum pH was 6.0, with a removal rate of 0.063–mg NO₃^?-N L^?1 h^?1     

Determination of specific oxygen uptake rate of Photorhabdus luminescens during submerged culture in lab scale bioreactor

Photorhabdus luminescens, a bacterial symbiont of entomoparasitic nematodes, was cultured in a 10 L bioreactor. Cellular density and bioluminescence were recorded and volumetric oxygen transfer coefficient (k_La) and specific oxygen transfer rates were determined during the batch process. Exponential phase of the bacterium lasted for 20 h, showing a maximum specific growth rate of 0.339 h^?1 in a defined medium. Bioluminescence peaked within 21h, and was maintained until the end of the batch process (48 h). The specific oxygen uptake rate (SOUR) was high during both lag and early exponential phase, and eventually reached a stable value of 0.33 mmol g^?1 h^?1 during stationary phase. Maintenance of 200 rpm agitation and 1.4 volume of air per volume of medium per minute (vvm) aeration, gave rise to a k_La value of 39.5 h^?1. This k_La value was sufficient to meet the oxygen demand of 14.4 g L^?1 (DCW) biomass. This research is particularly relevant since there are no reports available on SOURs of symbiotic bacteria or their nematode partners. The insight gained through this study will be useful during the development of a submerged monoxenic culture of Heterorhabditis bacteriophora and its symbiotic bacterium P. luminescens in bioreactors.     

Syngas fermentation to biofuel: Evaluation of carbon monoxide mass transfer coefficient (kLa) in different reactor configurations

Lignocellulosic biomass such as agri‐residues, agri‐processing by‐products, and energy crops do not compete with food and feed, and is considered to be the ideal renewable feedstocks for biofuel production. Gasification of biomass produces synthesis gas (syngas), a mixture primarily consisting of CO and H₂. The produced syngas can be converted to ethanol by anaerobic microbial catalysts especially acetogenic bacteria such as various clostridia species.One of the major drawbacks associated with syngas fermentation is the mass transfer limitation of these sparingly soluble gases in the aqueous phase. One way of addressing this issue is the improvement in reactor design to achieve a higher volumetric mass transfer coefficient (k_La). In this study, different reactor configurations such as a column diffuser, a 20‐μm bulb diffuser, gas sparger, gas sparger with mechanical mixing, air‐lift reactor combined with a 20‐μm bulb diffuser, air‐lift reactor combined with a single gas entry point, and a submerged composite hollow fiber membrane (CHFM) module were employed to examine the k_La values. The k_La values reported in this study ranged from 0.4 to 91.08 h^?1. The highest k_La of 91.08 h^?1 was obtained in the air‐lift reactor combined with a 20‐μm bulb diffuser, whereas the reactor with the CHFM showed the lowest k_La of 0.4 h^?1. By considering both the k_La value and the statistical significance of each configuration, the air‐lift reactor combined with a 20‐μm bulb diffuser was found to be the ideal reactor configuration for carbon monoxide mass transfer in an aqueous phase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Continuous multistep versus fed‐batch production of ethanol and xylitol in a simulated medium of sugarcane bagasse hydrolyzates

Co‐cultures for simultaneous production of ethanol and xylitol were studied under different operation bioreactor modes using Candida tropicalis IEC5‐ITV and Saccharomyces cerevisiae ITV01‐RD in a simulated medium of sugarcane bagasse hydrolyzates. Xylitol and ethanol tolerance by S. cerevisiae and C. tropicalis, respectively, was evaluated. The results showed that C. tropicalis was sensitive to ethanol concentrations up to 30 g/L, while xylitol had no effect on S. cerevisiae viability and metabolism. The best condition found for simultaneous culture was S. cerevisiae co‐culture and C. tropicalis sequential cultivation at 24 h. Under these conditions, productivity and yield for ethanol were Q_EtOH = 0.72 g L^?1 h^?1 and Y_EtOH/s = 0.37 g/g, and for xylitol, Q_XylOH = 0.10 g L^?1 h^?1 and Y_XylOH/S = 0.31 g/g, respectively; using fed‐batch culture, the results were Q_EtOH = 0.87 g L^?1 h^?1 and Y_EtOH/s = 0.44 g L^?1 h^?1, and Q_EtOH = 0.27 g L^?1 h^?1 and Y_EtOH/s = 0.57 g/g, respectively. Maximum volumetric productivity in continuous multistep cultures of ethanol and xylitol was at dilution rates of 0.131 and 0.074 h^?1, respectively. Continuous multistep production, Q_EtOH increased up to 50% more than in fed‐batch culture, even though xylitol yield remained unchanged.     

Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient

Aims: To improve the yield and productivity of docosahexaenoic acid (DHA) by Schizochytrium sp. in terms of the analysis of microbial physiology. Methods and Results: A two‐stage oxygen supply control strategy, aimed at achieving high concentration and high productivity of DHA, was proposed. At the first 40 h, K_La was controlled at 150·1 h^?1 to obtain high μ for cell growth, subsequently K_La was controlled at 88·5 h^?1 to maintain high q_p for high DHA accumulation. Finally, the maximum lipid, DHA content and DHA productivity reached 46·6, 17·7 g l^?1 and 111 mg l^?1 h^?1, which were 43·83%, 63·88% and 32·14% over the best results controlled by constant K_La. Conclusions: This paper described a two‐stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizochytrium sp. Significance and Impact of the study: This study showed the advantage of two‐stage control strategy in terms of microbial physiology. As K_La is a scaling‐up parameter, the idea developed in this paper could be scaled‐up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.     

Fish as sources and sinks of nutrients in lakes

1. The release of total phosphorus (TP) and nitrogen (N in ammonium) was measured for the five most abundant fish species (>85% of biomass) in Mouse and Ranger Lakes, two biomanipulated, oligotrophic lakes in Ontario. 2. The specific release rate of both nutrients was significantly related to fish mass; log₁₀ TP release rate (μg h^?1) = 0.793 (±0.109) [log₁₀ wet mass (g)] + 0.7817 (±0.145), and log₁₀ N release rate (μg h^?1) = 0.6946 (±0.079) [log₁₀wet mass (g)] + 1.7481 (±0.108). 3. When fish nutrient release was standardized for abundance (all populations, 1993–95) and epilimnetic volume, fish were estimated to contribute 0.083 (±0.061) μg TP L^?1 day^?1, and 0.41 (±0.17) μg N L^?1 day^?1 in Mouse L., and 0.062 (±0.020) μg TP L^?1 day^?1 and 0.31 (±0.08) μg N L^?1 day^?1 in Ranger L. 4. In comparison, concurrent rates of total planktonic P regeneration were 1.02 (±0.45) μg L^?1 day^?1 (Mouse L.) and 0.85 (±0.19) μg L^?1 day^?1 (Ranger L.). Fish represented 8% of planktonic P release in Mouse L. and 7% in Ranger L. 5. Fish dry mass had mean elemental body compositions of 39.3% carbon, 10.9% nitrogen, and 4.0% phosphorus (all fish combined), with a mean molar C : N : P ratio of 27 : 6 : 1. This comprised about 55% and 23% of the total epilimnetic particulate P and N respectively. 6. Turnover times of P and N in fish were approximately 103 and 48 days respectively. In comparison, planktonic turnover times of particulate P in Mouse and Ranger Lakes were 4.3 and 4.4 days respectively. Given their high P content and low turnover rates, fish appear to be important P sinks in lakes.     

Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland)

The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to ～3.4 μM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 μM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 × 10⁷ cells ml^?1). Incubation experiments with ¹⁵N‐nitrite revealed nitrogen loss occurring in the chemocline through denitrification (～3 nM N h^?1). At the same depth, incubations experiments with ¹⁵N₂‐ and ¹³C_DIC‐labelled bicarbonate, indicated substantial N₂ fixation (31.7–42.1 pM h^?1) and inorganic carbon assimilation (40–85 nM h^?1). Catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N₂ fixers, with the highest expression levels right at the chemocline. The majority of N₂ fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization‐Secondary Ion Mass Spectroscopy (HISH‐SIMS), we could for the first time directly link Chlorobium to N₂ fixation in the environment. Moreover, our results show that N₂ fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.     

Batch and fed-batch fermentation of optically pure D (-) lactic acid from Kodo millet (Paspalum scrobiculatum) bran residue hydrolysate: growth and inhibition kinetic modeling

Abstract

A low-cost Kodo millet bran residue was utilized as feedstock for the production of D (?) lactic acid (DLA) using Lactobacillus delbrueckii NBRC3202 under anaerobic condition. Data culled from a series of batch fermentation processes with different initial Kodo millet bran residue hydrolysate (KMBRH) and DLA concentrations were used for kinetic model development. Both simulated and experimental data were in good agreement for cell growth, KMBRH utilization, and DLA formation. The values of kinetic constants specific growth rate, (μ_m = 0.17?h^?1); growth (α_P = 0.96?g.g^?1) and non-growth (β_P = 1.19?g.g^?1.h^?1) associated constant for DLA production and the maximum specific KMBRH utilization rate, (q_{G, max} = 1.18?g.g^?1.h^?1) were in good agreement with the literature reports. Kinetic analysis elucidated that L. delbrueckii growth was predominantly influenced by KMBRH limitation and highly sensitive to DLA inhibition. Fed-batch fermentation studies demonstrated the existence of substrate and product inhibition paving the scope for process intensification.     

Steps Toward High‐Performance PLA: Economical Production of d‐Lactate Enabled by a Newly Isolated Sporolactobacillus terrae Strain

Optically pure d ‐lactate production has received much attention for its critical role in high‐performance polylactic acid production. However, the current technology can hardly meet the comprehensive demand of industrialization on final titer, productivity, optical purity, and raw material costs. Here, an efficient d ‐lactate producer strain, Sporolactobacillus terrae (S. terrae) HKM‐1, is isolated for d ‐lactate production. The strain HKM‐1 shows extremely high d ‐lactate fermentative capability by using peanut meal, soybean meal, or corn steep liquor powder as a sole nitrogen source; the final titers (205.7 g L^?1, 218.9 g L^?1, and 193.9 g L^?1, respectively) and productivities (5.56 g L^?1 h^?1, 5.34 g L^?1 h^?1, and 3.73 g L^?1 h^?1, respectively) of d ‐lactate reached the highest level ever reported. A comparative genomic analysis between S. terrae HKM‐1 and previously reported d ‐lactate high‐producing Sporolactobacillus inulinus (S. inulinus) CASD is conducted. The results show that many unrelated genetic features may contribute to the superior performance in d ‐lactate production of S. terrae HKM‐1. This d ‐lactate producer HKM‐1, along with its fermentation process, is promising for sustainable d ‐lactate production by using agro‐industrial wastes.     

Kinetic Modeling for Simultaneous Organic Carbon Oxidation,Nitrification, and Denitrification of Abattoir Wastewater in Sequencing Batch Reactor

A laboratory-scale study was conducted in a 20.0-L sequencing batch reactor (SBR) to explore the feasibility of simultaneous removal of organic carbon and nitrogen from abattoir wastewater. The reactor was operated under three different combinations of aerobic-anoxic sequence, viz., (4+4), (5+3), and (5+4) h of total react period, with influent soluble chemical oxygen demand (SCOD) and ammonia nitrogen (NH₄⁺-N) level of 2200 ± 50 and 125 ± 5 mg L^?1, respectively. In (5+4) h cycle, a maximum 90.27% of ammonia reduction corresponding to initial NH₄⁺-N value of 122.25 mg L^?1 and 91.36% of organic carbon removal corresponding to initial SCOD value of 2215.25 mg L^?1 have been achieved, respectively. The biokinetic parameters such as yield coefficient (Y), endogenous decay constant (k_d), and half-velocity constant (K_s) were also determined to improve the design and operation of package effluent treatment plants comprising SBR units. The specific denitrification rate (q_DN) during anoxic condition was estimated as 6.135 mg N/g mixed liquor volatile suspended solid (MLVSS)·h on 4-h average contact period. The value of Y, k_d and K_s for carbon oxidation and nitrification were found to be in the range of 0.6225–0.6952 mg VSS/mg SCOD, 0.0481–0.0588 day^?1, and 306.56–320.51 mg L^?1, and 0.2461–0.2541 mg VSS/mg NH₄⁺-N, 0.0324–0.0565 day^?1, and 38.28–50.08 mg L^?1, respectively, for different combinations of react periods.     

Butanol production from concentrated lactose/whey permeate: Use of pervaporation membrane to recover and concentrate product

In these studies, butanol (acetone butanol ethanol or ABE) was produced from concentrated lactose/whey permeate containing 211 g L^?1 lactose. Fermentation of such a highly concentrated lactose solution was possible due to simultaneous product removal using a pervaporation membrane. In this system, a productivity of 0.43 g L^?1 h^?1 was obtained which is 307 % of that achieved in a non-product removal batch reactor (0.14 g L^?1 h^?1) where approximately 60 g L^?1 whey permeate lactose was fermented. The productivity obtained in this system is much higher than that achieved in other product removal systems (perstraction 0.21 g L^?1 h^?1 and gas stripping 0.32 g L^?1 h^?1). This membrane was also used to concentrate butanol from approximately 2.50 g L^?1 in the reactor to 755 g L^?1. Using this membrane, ABE selectivities and fluxes of 24.4–44.3 and 0.57–4.05 g m^?2 h^?1 were obtained, respectively. Pervaporation restricts removal of water from the reaction mixture thus requiring significantly less energy for product recovery when compared to gas stripping.     

Quantitative aspects of oxygen and carbon dioxide exchange through the lungs in Ocypode ceratophthalmus (Crustacea: Decapoda) during rest and exercise in water and air

Ghost crabs Ocypode ceratophthalmus were exercised in air and water to measure CO₂ and O₂ exchange rates using the method of instantaneous measurements of oxygen consumption rate (MO₂) where applicable. Average heart rate increased from 100 to nearly 400 pulses per minute after five minutes of exercise on a treadmill at a run rate of 0.133 m s^?1. It took less than a minute for oxygen taken up through the lung epithelium from the air inside the branchial cavity to reach the maximal oxygen consumption rate of 26.1 mmol O₂ kg^?1 h^?1. Resting MO₂ was 4.06 mmol O₂ kg^?1 h^?1 in air, but decreased to 3.37 mmol O₂ kg^?1 h^?1 in seawater. Radioactive CO₂ from injected l-lactate is released linearly by the lung. The percent accumulated 14-CO₂ in exhaled air, plotted against time, intersects zero time on the x -axis, indicating rapid gas exchange at the lung surface. The P ₅₀ values for native haemocyanin of 4.89 mm Hg before exercise, and 8.99 mm Hg after exercise, are typical of a high-affinity haemocyanin usually associated with terrestrial crabs. The current notion that Ocypode ceratophthalmus drown when submerged in seawater was not substantiated by our experiments. MO₂ in seawater increased from 3.37 mmol O₂ kg^?1 h^?1 for resting crabs to 5.72 mmol O₂ kg^?1 h^?1 during exercise. When submerged by wave-seawater in the natural environment and during exercise in respirometer-seawater O. ceratophthalmus do not swim but, having a specific density of 1.044, float nearly weightless with a minimum of body movements.