New sensor allowing continuous water activity measurement of submerged or solid-substrate fermentations

Water activity of the substrate is an important and acute factor for growth as well as for metabolic production of microorganisms or for biocatalyst systems. A sensor has been designed in order to control this parameter on-line during submerged and solid-substrate fermentations. This sterilizable sensor allows the measurement of the relative humidity of the atmosphere in a small chamber by means of a capacitive element separated from the medium by a thin ethylenepolytetrafluoride membrane. For high a(w) values (>0.90) a sequential circulation of a dried gas prevents the sensor saturation. Measurements are rapid and accurate, and control of the water activity of a fermentation medium has been carried out for 5 days using this sensor.     

Computer-aided control of water activity for lipase-catalyzed esterification in solvent-free systems

A computer system for on-line monitoring and control of the water activity (a(w)) in solvent-free media has been developed. The performance of this system was investigated by carrying out the lipase-catalyzed esterification of n-capric acid with n-decyl alcohol. A humidity sensor measured the relative humidity in the reactor headspace, which was then transmitted electrically to a digital computer that was used as a feedback controller. The water activity control was achieved by sparging either humidified air or dried air through the reaction medium at a flow rate determined by the digital feedback controller. The use of humid air and dry air for a(w) control made it possible to induce a larger a(w) gradient and thereby higher water transfer rate. As a result, the water activity quickly reached the desired a(w) values. We tested whether water activity in the reaction medium can be monitored by measuring relative humidity in the headspace. When the water activity in the liquid phase was determined from measurements of water content in the medium and compared to that measured directly with the humidity sensor, the a(w) in the reaction medium did not differ significantly from that in the headspace. This indicates that there is a near-equilibrium between the liquid medium and the exit air stream. Water activity was also successfully maintained close to the set point despite the massive production of water during the esterification process. Thus, the control system developed in this study is particularly useful for systems where large amounts of water are produced and where conventional methods make it difficult to control water activity as a result of a low water transfer rate. The effects that computer control of the water activity had on the reaction rate and yield were also examined. The reaction yield was significantly improved with water activity control. The conversions obtained at 28 h without and those with water activity control were 70% and 96%, respectively. In addition, from the fact that the final yields increased with decreasing a(w), computer-aided water activity control was performed with a set-point change. By controlling a(w) at 0.55 during initial reaction phase, followed by a step change of a(w) from 0.55 to 0 after 11 h of reaction, it was possible to enhance the final conversion to 100%.     

A water activity control system for enzymatic reactions in organic media

A water activity control system for enzymatic synthesis in organic media, for litre-scale reactors has been constructed. Water activity, a(w), is a key factor when using enzymes in non-conventional media and the optimum value varies for different enzymes. The control system consists of a water activity sensor in the headspace of a jacketed glass reactor (equipped with narrow steel tubes to introduce air), gas-washing bottles containing blue silica gel (a(w)=0) and water (a(w)=1), a PC to monitor water activity and a programmable logic controller (PLC) to control the water activity. The system was evaluated by adjusting water activity in the medium, with a deviation from the set point of less than +/-0.05. Synthesis of cetyl palmitate, under controlled water activity and catalysed by two different lipase preparations, namely, Novozym 435 (immobilised Candida antarctica lipase B) and immobilised Candida rugosa lipase, were also performed. Novozym 435 catalyses reactions very well at extremely low water activity while C. rugosa lipase shows low activity for a(w)<0.5.     

Investigation of the biological activity of solutions activated electrochemically in a membrane electrolyzer

The biological activity of the catholyte and anolyte of double distilled water was studied in experiments on the germination of wheat grains in the period from March to May. The activity of the solutions, which was characterized by a growth index, was high early in this period, then decreased almost to zero in the middle of the period, and then increased to about the initial value by the end of the period. Throughout, the efficiency of the anolyte of double distilled water generally exceeded the efficiency of the catholyte. Early and late in the period, the stimulatory effect of the anolyte exceeded that of the catholyte by a factor of 5–5.5. The changes in the biological activity of the catholyte and anolyte of double distilled water were also compared with the changes in the biological activity of the catholyte of nutrient medium M9. The stimulatory effect of the catholyte of the nutrient medium was evaluated from the change in the growth of E. coli cells. Early in the period at a cultivation temperature of 20°C, the stimulatory effect determined from the increase in the optical density of the cell suspension in the experiment with respect to a reference value was 55–60%. Next, the stimulatory effect decreased almost to zero in the middle of the period and increased to approximately initial value by the end of the period. It was assumed that the physicochemical mechanisms of action of the catholyte and anolyte of double distilled water on the wheat seed germination and of the catholyte of the nutrient medium on E. coli cell growth are of different nature.     

Effect of Water Activities of Heating and Recovery Media on Apparent Heat Resistance of Bacillus cereus Spores

Spores of Bacillus cereus were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores. The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors. Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity. As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose. In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values. This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose. When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the efficiency of depressors was also less pronounced. It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.     

Water activity: a fundamental parameter of aroma production by microorganisms

Summary The water activity of the medium, which is analogous to osmotic pressure in liquid medium, is a fundamental parameter for the mass transfer of water and solutes across the cell membrane. The control of this parameter could be used to modify the metabolic production or excretion of a microorganism, as demonstrated in this work for aroma production by a fungus and a yeast. Offprint requests to: P. Gervais     

Modulation of protease specificity by a change in the enzyme microenvironment. Selectivity modification on a model substrate, purified soluble proteins and gluten

Subtilisin BPN' activity on a synthetic substrate is found to decrease with the concentration of soluble additives such as sugars and polyols, the catalytic efficiency of the enzyme being related to the water activity in the reaction medium. Limited hydrolysis of B chain of insulin is followed and the cleavage priority determined. When carried out in glycerol-containing medium, both enzyme catalytic behaviour and specificity are perturbed; a different cleavage order and a selectivity restriction are observed. The experiments were generalised to purified proteins and to an insoluble protein complex. The hydrolysis kinetics of purified gliadins by pepsin and of gluten by a Bacillus neutral protease are modulated in presence of water activity depressors. Glycerol is able to increase both pepsin efficiency and gluten protein solubility. The hydrolysis order is affected by water-structuring molecules in the enzyme microenvironment and new peptides appear whatever the size and initial solubility of the substrate.     

Reaction Kinetics of Immobilized α-Chymotrypsin in Organic Media 1. Influence at Solvent Polarity

Esterification of N-acetyl phenylalanine with ethanol catalyzed by immobilized α-chymotrypsin (EC 3.4.21.1) was studied in various reaction media. The effect of reaction medium polarity on enzymatic activity as well as equilibrium yield was measured. The reaction rate increased with increasing amounts of added water, reaching an optimum corresponding to water saturation of the reaction medium. Further additions of water resulted in decreased activity. Bell-shaped activity profiles were obtained for all reaction media tested. Reaction media consisting of pure solvents and of mixtures of solvents were used. The enzymatic activity and the equilibrium yield increased with decreased polarity of the medium. Maximum activity was found in a reaction medium consisting of 80% diisopropyl ether and 20% heptane. The enzymatic activity obtained at optimal water additions in the different solvents and solvents mixtures could be correlated to the solubility of water and the log P of the medium. The equilibrium yield of the reaction was much more closely correlated to the solubility of water than the log P. Much lower enzymatic activity was obtained when solvent mixtures producing water-miscible media were created, than in mixtures producing water-immiscible media, such as mixtures of acetonitrile and diisopropyl ether. The equilibrium yield could be increased by decreasing the water content in the reaction medium, which reduced the water activity.     

Alcohols as enantioselective inhibitors in a lipase catalysed esterification of a chiral acyl donor

Summary Increased reaction rates and increased enantioselectivities were observed with decreased concentrations of n-alkanols when resolving 2-methyldecanoic acid by esterification catalysed by immobilised lipase from Candida rugosa at controlled water activities in cyclohexane. The enantioselectivity was found to be independent of the water activity in the reaction medium at the n-heptanol concentrations investigated. However, when n-decanol was used as the acyl acceptor, not only the alcohol concentration but also the water activity in the reaction medium, influenced the enantioselectivity. The results obtained showed that the low enantioselectivity seen at a high alcohol concentration could be explained by the alcohol influencing the apparent V _max ^S and V _max ^R differently.     

Influence of the organic solvents on the activity in water and the conformation of Candida rugosa lipase: Description of a lipase-activating pretreatment

The influence on lipase activity in water of a pretreatment on Candida rugosa lipase using water miscible and immiscible solvents was studied. The lipase activity in the hydrolysis of esteric substrates in aqueous media increases when the lipase was previously treated with various nearly anhydrous organic media. This activation, which was irreversible, was higher for longer pretreatment times. It was dependent on the pretreatment medium (water activity and solvent used). A relation between variations in the emission intensity and the activities of treated and untreated lipases was found. Activating pretreatment did not shift the peak of fluorescence emission but gave rise to variations in the secondary protein structure by increasing the helical nature. A similar increment in the hydrolysis rate in water can be obtained with the addition of an appropriate amount of solvent (acetonitrile or n-heptane) to the aqueous reaction medium.     

Sugar ester synthesis by a mycelium-bound Mucor circinelloides lipase in a micro-reactor equipped with water activity sensor

The mycelium-bound Mucor circinelloides lipase was used for the synthesis of esters of saccharides and fatty acids in 37 ml reactor equipped with magnetic stirrer and water activity sensor. Either di-n-pentyl ether or the mixture of di-n-pentyl and petroleum ethers were applied as reaction media. Water activity sensor provided on-line monitoring of this parameter and control of continuous processes of ester synthesis. It was found that two natural antioxidants, i.e. carotene and astaxanthin activated this lipase in organic solvents that could be beneficial for the synthesis of esters of compounds sensitive to oxidation, e.g. polyunsaturated fatty acids.     

Influence of water activity on the ice-nucleating activity of Pseudomonas syringae

Pseudomonas syringae is known as a biological ice-nucleating agent. The bacterium has the unusual property of increasing the temperature at which water freezes by a few degrees. However, the ice-nucleating activity (INA) always remains lower for in vitro cultivated cells, than for cells grown in planta. We examined the effects of the hydrophobic environment and of water availability, on the in vitro growth and INA of P. syringae. The hydrophobic environment was modified by addition of fatty acids, vegetable oils or silicone oil to the culture medium. Addition of olive oil (1%), or traces of silicone oil in the culture medium had a positive effect upon the expression of INA. Variations in water activity from 0.990 to 0.988 by addition of sugar beet fibres or sodium chloride in the culture medium were followed by an increase in INA. This study suggested that control of the medium’s water activity must be considered as an important parameter for optimization of INA in P. syringae. Received 16 June 1998/ Accepted in revised form 02 September 1999     

Effect of the sugar-beet pulp water activity on the solid-state culture of Trichoderma viride TS

Summary The growth and the sporulation of Trichoderma viride TS in relation to water activity (a _w) of sugar-beet pulp medium was studied. It was found that the maximum growth, monitored by protein production, substrate utilization and pH alteration, appeared at a _w=0.990–0.992. Optimal water activity of the medium for sporogenesis was 0.980. It was observed that both physiological phenomena appeared in narrow ranges of water activity which caused the rigorous a _w control in solid-state fermentation to be postulated.     

Opercular differential pressure as a predictor of metabolic oxygen demand in the starry flounder

The feasibility of using a differential pressure sensor connected to an acoustic telemetry device to monitor opercular activity as a correlate of oxygen consumption was investigated. Four starry flounders Platichthys stellatus were fitted with a miniature differential pressure sensor mounted close to the operculum. A cannula was connected to the sensor and inserted under the operculum, inside the branchial cavity. Measurements of oxygen consumption and opercular activity were carried out over a broad range of metabolic activity, from the post‐surgery stress (high metabolic rate) to routine metabolic rate the following day. Relationships between differential pressure changes (rate and amplitude) were highly correlated with oxygen consumption ( r ² = 0·74 and 0·60 respectively). The results indicate that monitoring opercular activity offers an alternative method for measuring aerobic metabolism in free‐swimming fishes in nature.     

Molecular oxygen regulates the enzymatic activity of a heme-containing diguanylate cyclase (HemDGC) for the synthesis of cyclic di-GMP

We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O₂, but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O₂ sensing responsible for the regulation of the enzymatic activity.     

Working at controlled water activity in a continuous process: the gas/solid system as a solution

Fusarium solani cutinase and Candida cylindracea lipase were used to catalyze a transesterification reaction in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrate and removed reaction products simultaneously. Different conditions of immobilization were used and compared to the results obtained with a nonsupported enzyme. The enzymatic activity was found to be highly dependent of a key parameter: water activity (a(w)). Biocatalyst stability was greatly influenced by water activity and the choice of immobilization technique for the enzymatic material. For free and adsorbed enzymes, water requirements exhibited optima which corresponded to the complete hydration coverage of the protein. These optima presented a good correlation with the isotherm sorption curves obtained for the different preparations. In this work are reported the results concerning the possibility of using a continuous system able to operate at controlled water activity in a heterogeneous medium. Lipolytic enzyme in such a system appears to be a new process for the biotransformation of volatile esters. (c) 1995 John Wiley & Sons, Inc.     

Effect of environmental acidity and alkalinity on the physiology of Tilapia mossambica during acclimation

The pattern of carbohydrate metabolism in the muscle of the freshwater fish, Tilapia mossambica (Peters) varied according to the pH of the environmental medium. On acclimation to a more acidic water, the white muscle showed an elevated glycogen content with suppressed glycolysis. In contrast, the muscle tissue showed an accelerated glycolytic pathway with the accumulation of metabotic acids on acclimation to a basic water. In an acidic medium phosphorylase activity was inhibited with an elevated LDH activity, while the reverse pattern was observed in an alkaline medium. The oxidative pathway was elevated in both. Acclimation to acidic and basic environmental waters leads to an adaptive compensatory mechanisms providing increased resistance capacity to the fish under pH stress.     

Predicting the Performance of Surface Plasmon Resonance Sensors Based on Anisotropic Substrates

The aim is to investigate how the sensing features of a surface plasmon resonance sensor based on the Kretschmann configuration are affected when the optical substrate is an anisotropic medium. The investigation considers the use of two different uniaxial anisotropic crystals, one made of barium titanate and another made of lithium niobate, as the optical substrate. It also considered that the sensor operates in the angular interrogation mode at the gold–water interface. The Fresnel equations and the finite element method were employed to determine the sensing features. The present study revealed that both formulations provide almost identical results for the resonance angle (difference less than 1%). On the other hand, the two formulations provide results with significant differences for additional features like the sensitivity and the full width at half maximum.