Animal and worker exposure to dust and biological particles in animal care houses

An aerobiological study was performed to evaluate the potential exposure of animals and workers to dust constituents generated during routine animal house work. Different rooms of air conditioned (A, control) and passively ventilated (B, non-air conditioned) animal facilities were sampled, in order to evaluate total airborne culturable fungi and bacteria, fungal spore concentrations and particle levels. Airborne room particles were analyzed gravimetrically and for endotoxin content. All parameters, except for culturable fungi, were higher in facility B and statistically significant, with respect to those from the control facility A. Median values for airborne particle concentration, endotoxin and fungal spores in facility B were: 115 µg m^–3, 25 EU m^–3, and 2173 spores m^–3, respectively. Median values for facility A were: 66 µg m^–3, 9 EU m^–3, and 248 fungal spores m^–3. Broncheoalveolar lavage from rats kept in the rat room of B, presented median concentrations of total cells and lactate dehydrogenase, higher than those found in the control facility (4.4 × 10⁵ vs. 1.1 × 10⁵ and 2.7 UmL^-1 vs. 0.39 UmL^–1, respectively). Values of total and biological particles of both facilities, as well as the time spent in different rooms, showed that worker exposure was higher during cage washing. It was especially high in the passively ventilated facility (airborne particles 686 µg m^–3 3.5 h^–1 vs. 976 µg m^–3 3.5 h^–1, endotoxin 70 EU m^–3 3.5 h^–1 vs. 108 EU m^–3 3.5 h^–1). The type of basidiospores and ascospores found, as well as the significant correlation between particle levels and endotoxin contents suggests that wood chip bedding disturbance during cage washing is an important source for airborne biological particles. The changes in broncheoalveolar lavage components found in rats from these facilities and previously reported changes in pro-inflammatory cellular responses found in workers, indicate that these relatively low levels of exposure are enough to induce a biological response. Studies considering the composition of mixed organic dusts, would be needed to reevaluate current occupational standards.     

Production of xanthan by in-flow cultures of Xanthomonas campestris

The kinetics of xanthan formation in Xanthomonas campestris continuous and fed-batch fermentations was studied along with metabolic changes due to growth rate variation. A maximum growth rate within the range 0.11–0.12 h^–1 was obtained from the continuous culture data in defined medium, producing xanthan at rates up to 0.36 g l^–1 h^–1 corresponding to a maximum 67% glucose conversion at a dilution rate (D) of 0.05 h^–1. Comparatively, fed-batch cultivation was more efficient, producing maximum xanthan at 0.75 g l^–1 h^–1 and 63% glucose conversion at 0.1 h^–1. When reaching D=0.062 h^–1 in continuous cultures, a change was observed and the values of the specific rate of substrate consumption shifted, initiating an uncoupled growth region expressing a lack of balance of the catabolic and anabolic reactions. The deviation was not accompanied by a change in specific xanthan production indicating that xanthan metabolism was not affected by D. For fed-batch-grown X. campestris cells within the range D=0.03–0.1 h^–1, both metabolic parameters changed linearly with the growth rate showing a wide region coupled to growth. Outside that range, glucose accumulated and the specific xanthan production dropped, suggesting substrate inhibition. Correspondence to: J. C. Roseiro     

Continuous production of poly(3-hydroxybutyrate-co-3-hyhroxyvalerate) byAlcaligenes eutrophus

Summary Copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced in a continuous culture ofAlcaligenes eutrophus with 17.5 gl ^–1 of fructose and 2.5gl ^–1 of pentanoic acid in the feed. The P(3HB-co-3HV) productivity was maximal at a dilution rate of 0.17h^–1 and yielded 0.31gl ^–1h^–1 under an ammonium-limited condition. The 3HV content in copolymers increased from 11 to 79 mol% as the dilution rate of cells was increased from 0.06 to 0.32h^–1.     

Degradation of sodium anthraquinone sulphonate by free and immobilized bacterial cultures

Aerobic biodegradation of a xenobiotic recalcitrant compound sodium anthraquinone-2-sulphonate (SAS), was investigated using as an inoculum a mixed microbial culture, which was activated sludge from industrial and domestic waste-water treatment plants. The difference in SAS degradation was examined using two main systems: (1) suspended cells and (2) immobilized cells, both in batch and in continuous culture. In the suspended cell system, under continuous culture conditions using SAS as a unique source of carbon and energy, it was possible to degrade about 95% of this substrate after 6 days. Maximal SAS removal rates in the suspended-cell system were 593 mg SAS l^–1 h^–1 and 88.7 mg SAS l^–1 h^–1 for dilution rates (D) of 0.05 h^–1 and 0.075 h^–1, respectively. In the immobilized-cell system, almost all SAS was degraded in 6 days and the maximal removal rate reached 88.7 mg SAS l^–1 h^–1 at D=0.05 h^–1. Application of a continuous-flow enrichment procedure resulted in selection of several kinds of micro-organisms and led to a progressive elimination of some species of Aeromonas. A stable microbial community of 11 strains has been established and characterized at D=0.075 h^–1. Most of them were Gram-negative and belonged to the genus Pseudomonas.     

Effect of the dilution rate on the biomass yield ofBacillus thuringiensis and determination of its rate coefficients under steady-state conditions

Depending on the biomass yield on glucose and the cell morphology ofBacillus thuringiensis, three different metabolic states were observed in continuous culture. At dilution rates between 0.18 h^–1 and 0.31 h^–1 vegetative cells, sporulating bacteria and spores coexisted, while glucose and amino acids were consumed. Only vegetative cells were observed at dilution rates between 0.42 h^–1 and 0.47 h^–1 and glucose was used as the main carbon and energy source. AtD = 0.50 h^–1 the biomass yield on glucose decreases sharply. To define better the specific growth rate range in which the microorganism uses mainly glucose, a dilution rate of 0.25–0.45 h^–1 was studied. The experimental data could be adjusted to a Monod model and the following rate coefficients and growth yields were determined: maximum specific growth rate 0.54 h^–1, saturation constant 0.56 mg glucose ml^–1, biomass growth yields 0.43 g cells (g glucose)^–1, and 0.76 g cells (g oxygen)^–1, and maintenance coefficients 0.065 g glucose (g cells)^–1 h^–1 and 0.039 g oxygen (g cells)^–1 h^–1.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Lactic acid productivity of a continuous culture of Lactobacillus delbreuckii

Summary Maximum volumetric productivities of biomass (1.40 gl^–1h^–1) and lactic acid (8.93 gl^–1h^–1) for a continuous culture ofLactobacillus delbreuckii occurred between dilution rates 0.35h^–1 and 0.40h^–1. All major nutrients were in excess in these cultures. Glucose utilisation was complete at dilution rates of 0.1h^–1 and lower. Product and biomass yields were constant in the dilution rate range studied (0.05h^–1 to 0.50h^–1).     

n-3 Polyunsaturated fatty acid productivity of the marine microalgaIsochrysis galbana. Growth conditions and phenotypic selection

Two set of isolates were obtained in an isolation/selection programme to select eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) rich strains ofIsochrysis galbana. EPA content was improved up to an average of 5.3% (d.wt) for the second set of isolates. On the other hand, with the aeration rate, pH and irradiance kept at low levels, the growth rate was slow and EPA synthesis was enhanced, but productivity increased when growth rates were maximum. A model relating steady-state dilution rates in chemostat cultures ofI. galbana to internal average irradiance is proposed. The greatest productivities were obtained between 0.0295 h^–1 and 0.0355 h^–1 with 300 mg m^–3 h^–1 for EPA and 130 mg m^–3 h^–1 for DHA.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

The Effects of Food Concentration and Quality on the Feeding Rates of Three Size Classes of the Greenshell<Superscript>TM</Superscript> Mussel, <Emphasis Type="Italic">Perna canaliculus</Emphasis>

In order to calibrate carrying capacity models, investigations were conducted into the effects of food concentration and food quality on the feeding rates of small (25–50 mm), medium (60–85 mm) and large (90–115 mm) Greenshell mussels (Perna canaliculus). Experimental diets varying from 3.3 to 6.0 μg l⁻¹ chlorophyll a concentration and 12–25% organic content were fed to mussels housed in individual flow through chambers. Not surprisingly, this study found that the main factor affecting feeding rates is mussel size. Small mussels were observed to maintain a constant filtration rate of approximately 20 mg h⁻¹ irrespective of food concentration or quality, whereas mussels of greater than 60 mm length had more variable filtration rates between 30 and 80 mg h⁻¹. The filtration rates of these large mussels were also observed to increase positively with organic content, and showed no sign of levelling out, even at the highest organic content tested (25%). Highest rejection rates (50–70 mg h⁻¹) were observed when the organic content of the available seston was low, suggesting that P. canaliculus are able to selectively reject organic material, thereby organically enriching their diet. It appears that the organic content of the seston is the primary determinant of the net efficiency with which food is selected from the available seston by the mussel. The present study shows that P. canaliculus of all sizes are capable of adapting their feeding behaviour to compensate for changes in the food supply, which may occur over relatively short time periods, in the culture environment.     

Respiration of individual honeybee larvae in relation to age and ambient temperature

The CO₂ production of individual larvae of Apis mellifera carnica, which were incubated within their cells at a natural air humidity of 60–80%, was determined by an open-flow gas analyzer in relation to larval age and ambient temperature. In larvae incubated at 34 °C the amount of CO₂ produced appeared to fall only moderately from 3.89±1.57 µl mg^–1 h^–1 in 0.5-day-old larvae to 2.98±0.57 µl mg^–1 h^–1 in 3.5-day-old larvae. The decline was steeper up to an age of 5.5 days (0.95±1.15 µl mg^–1 h^–1). Our measurements show that the respiration and energy turnover of larvae younger than about 80 h is considerably lower (up to 35%) than expected from extrapolations of data determined in older larvae. The temperature dependency of CO₂ production was determined in 3.5-day-old larvae, which were incubated at temperatures varying from 18 to 38 °C in steps of 4 °C. The larvae generated 0.48±0.03 µl mg^–1 h^–1 CO₂ at 18 °C, and 3.97±0.50 µl mg^–1 h^–1 CO₂ at 38 °C. The temperature-dependent respiration rate was fitted to a logistic curve. We found that the inflection point of this curve (32.5 °C) is below the normal brood nest temperature (33–36 °C). The average Q₁₀ was 3.13, which is higher than in freshly emerged resting honeybees but similar to adult bees. This strong temperature dependency enables the bees to speed up brood development by achieving high temperatures. On the other hand, the results suggest that the strong temperature dependency forces the bees to maintain thermal homeostasis of the brood nest to avoid delayed brood development during periods of low temperature.Abbreviations m body mass - R rate of development or respiration - T_I inflexion point of a logistic (sigmoid) curve - T_L lethal temperature - T_O temperature of optimum (maximum) developmentCommunicated by G. Heldmaier     

Stable continuous constitutive expression of a heterologous protein in Saccharomyces cerevisiae without selection pressure

The stability of heterologous protein expression in Saccharomyces cerevisiae during continuous culture without selection for plasmid-containing cells was investigated. The protein chosen was the leech thrombin inhibitor desulphato-hirudin, which is tolerated well by S. cerevisiae when over-expressed. Expression was from a 2- derived multicopy vector containing a synthetic hirudin gene under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase derived GAPFL promoter. The behaviour of the system was studied at three dilution rates (D) corresponding to approximately 30% (0.06 h^–1), 60% (0.12 h^–1) and 90% (0.17 h^–1) of the estimated maximum D. The level of plasmid loss was low at all Ds, with only 5–10% plasmid-free cells observed at 75 generations. The plasmid was most stably maintained at the intermediate D of 0.12 h^–1, where the rate of loss was comparable to the loss of the native 2- plasmid. Hirudin expression was also highest at D=0.12 h^–1, possibly as a result of cell lysis at D=0.06 h^–1 and D=0.17 h^–1, leading to the release of vacuolar proteases and subsequent proteolysis of hirudin. Differences in expression levels were not a result of changes in plasmid copy number, which was in the range 40–60 throughout all three experiments. The high stability of this system at all Ds investigated shows that heterologous protein expression is not a burden to S. cerevisiae when the protein expressed is tolerated well. Correspondence to: M. Ibba     

Biooxidation of ferrous iron by immobilized Acidithiobacillus ferrooxidans in poly(vinyl alcohol) cryogel carriers

PVA-cryogels entrapping about 10⁹ cells of Acidithiobacillus ferrooxidans per ml of gel were prepared by freezing-thawing procedure, and the biooxidation of Fe²⁺ by immobilized cells was investigated in a 0.365 l packed-bed bioreactor. Fe²⁺ oxidation fits a plug-flow reaction model well. A maximum oxidation rate of 3.1 g Fe²⁺ l^–1 h^–1 was achieved at the dilution rate of 0.4 h^–1 or higher, while no obvious precipitate was determined at this time. In addition, cell-immobilized PVA-cryogels packed in bioreactor maintained their oxidative ability for more than two months under non-sterile conditions. Nomenclature: C _A0 – Concentration of Fe²⁺ in feed stream (g l^–1) C _A – Concentration of Fe^{2 +} in outlet stream (g l^{– 1}) D – Dilution rate of the packed-bed bioreactor (h^–1) F – Volumetric flow rate of iron solution (l h^–1) F _A0 – Mass flow rate of Fe²⁺ in the feed stream (g h^–1) K – Kinetic constant (l l^–1 h^–1) r _A – Oxidation rate of Fe²⁺ (g l^–1 h^–1) V – Volume of packed-bed bioreactor (l) X _A – Conversion ratio of Fe²⁺ (%)     

Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii

Two biofilters fed toluene-polluted air were inoculated with new fungal isolates of either Exophiala oligosperma or Paecilomyces variotii, while a third bioreactor was inoculated with a defined consortium composed of both fungi and a co-culture of a Pseudomonas strain and a Bacillus strain. Elimination capacities of 77 g m^–3 h^–1 and 55 g m^–3 h^–1 were reached in the fungal biofilters (with removal efficiencies exceeding 99%) in the case of, respectively, E. oligosperma and Paecilomyces variotii when feeding air with a relative humidity (RH) of 85%. The inoculated fungal strains remained the single dominant populations throughout the experiment. Conversely, in the biofilter inoculated with the bacterial–fungal consortium, the bacteria were gradually overgrown by the fungi, reaching a maximum elimination capacity around 77 g m^–3 h^–1. Determination of carbon dioxide concentrations both in batch assays and in biofiltration studies suggested the near complete mineralization of toluene. The non-linear toluene removal along the height of the biofilters resulted in local elimination capacities of up to 170 g m^–3 h^–1 and 94 g m^–3 h^–1 in the reactors inoculated, respectively, with E. oligosperma and P. variotii. Further studies with the most efficient strain, E. oligosperma, showed that the performance was highly dependent on the RH of the air and the pH of the nutrient solution. At a constant 85% RH, the maximum elimination capacity either dropped to 48.7 g m^–3 h^–1 or increased to 95.6 g m^–3 h^–1, respectively, when modifying the pH of the nutrient solution from 5.9 to either 4.5 or 7.5. The optimal conditions were 100% RH and pH 7.5, which allowed a maximum elimination capacity of 164.4 g m^–3 h^–1 under steady-state conditions, with near-complete toluene degradation.     

Daytime grazing and assimilation rates of planktonic copepods Acanthodiaptomus denticornis and Cyclops vicinus vicinus. Comparison of spatial and resource utilisation by rotifers and cladoceran communities in a eutrophic lake

The clearance rates of ¹⁴ carbon-labelled Chlamydomonas sp. by the copepodite and adult stages of the 2 dominant species of Copepoda were studied in eutrophic Lake Aydat. They varied from 30 to 3,007 µl ind^–1 h^–1 for Acanthodiaptomus denticornis (mean = 600 µl ind^–1 h^–1) and from 20 to 1,133 µl ind^–1 h^–1 for Cyclops vicinus (mean = 340 µl ind^–1 h^–1). Their maximum assimilation efficiencies were 58% and 50%, respectively. These populations collectively could consume the available food in 4 days during September (maximum daily grazing rate = 24%). Mean individual clearance rates could be ranked Acanthodiaptomus denticornis > Ceriodaphnia quadrangula > Chydorus sphaericus > Daphnia longispina > Cyclops vicinus vicinus > Bosmina longirostris > K. cochlearis > K. quadrata and Kellicottia longispina. Like cladocerans and rotifers, the copepods living in this eutrophic lake can feed at low oxygen concentrations.     

Deficiency of potassium but not phosphorus enhances root respiration

Root respiration of kohlrabi (Brassica oleraceavar. gongylodes) was measured non-destructivelyin vivo by infrared gas analysis of completeroot systems, using potted plants in sand culture andnutrient solutions, for six weeks under (a) nutrientsufficiency, (b) deficiency of all mineral nutrients,(c) potassium deficiency or (d) phosphorus deficiency.This was to study the adaptation to nutrient stress interms of changes in root growth, root respiration,assimilate allocation and energy requirement fornutrient uptake. Both deficiencies of phosphorus andpotassium increased the root:shoot-ratio. This wasattributed to the plants transferring a largerrelative proportion of assimilates to the roots thanto the shoots relative to nutrient-sufficient plants.Roots of nutrient-sufficient kohlrabi respired 1.7 or7.7 mg CO₂ h^–1 per g fresh or dry matter, respectively. However, potassiumdeficiency enhanced root respiration to 2.4 mgCO₂ h^–1 or 12.2 mg CO₂ h^–1 on a per g fresh or dry weight basis respectively. This originated from an additional2.6 mg glucose g^–1 dry matter h^–1 allocated to the roots and provided 50 Joule additional energy(150 versus 100 Joule g^–1 dry matter h^–1)which may become available for the proposedK⁺:H⁺ symporter for potassium uptake.     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

Anaerobic fermentation of Salmonella typhimurium with and without pyruvate carboxylase

A plasmid that expressed pyruvate carboxylase (PYC) from Rhizobium etli was introduced into Salmonella typhimurium LT2. Anaerobic fermentations of S. typhimurium with and without PYC were compared with glucose as a carbon source. The presence of PYC increased the succinate yield from glucose from 0.044 g g^–1 to 0.22 g g^–1, while the lactate yield decreased from 0.31 g g^–1 to 0.16 g g^–1. Metabolic flux calculations during the early growth phase indicate that under these growth conditions in the presence of PYC more carbon flows to oxaloacetate via pyruvate carboxylase than via phosphoenolpyruvate carboxylase. Also, under these growth and induction conditions, the presence of PYC diminished the cell growth rate from 0.34 h^–1 to 0.28 h^–1, the specific rate of ATP formation from 45 mmol l^–1 h^–1 to 27 mmol l^–1 h^–1, and the specific rate of glucose consumption from 17 mmol l^–1 h^–1 to 10 mmol l^–1 h^–1.     

Suspension feeding in Bithynia tentaculata (Prosobranchia,Bithyniidae), as affected by body size,food and temperature

The suspension feeding of Bithynia tentaculata was tested in laboratory experiments. The animals were fed in 1-1 aerated glass beakers, and filtration rates were calculated from changes in cell concentrations during the 6-h experiment. Temperature influenced the filtering rate, with minimum values of 5ml · ind^–1 · h^–1 at 5° C and maxima of 17.2 ml · ind^–1 · h^–1 at 18° C. Three food species of different size, motility and cell surface characteristics (Chlamydomonas reinhardii, Chlorella vulgaris and Chlorogonium elongatum) did not affect filtration rates. Suspension feeding increased with increasing food concentrations up to 12 nl · ml^–1, above which feeding rate was kept constant by lowering the filtering rates. Even the smallest animals tested (<4 mm body length) were found to be feeding on suspended food at a rate of 2.7 ml · ind^–1 · h^–1, and increasing rates up to 8.4 ml were found in the 6–7 mm size class. All size classes of Bithynia showed a circannual fluctuation of their filtration rates. The ecological consequences of Bithynia's ability to switch between two feeding modes, grazing and suspension feeding, are discussed.     

High efficiency styrene biodegradation in a biphasic organic/water continuous reactor

Styrene was degraded as sole source of carbon and energy by a selected bacterial community in a two-phase aqueous-organic medium (80%:20%, vol/vol). Silicone oil was used to solubilize styrene, which is sparingly soluble in water and to prevent its toxicity toward microorganisms. Preliminary studies with the mixed population in batch cultures indicate that the specific activity and the maximum growth rate at optimal 3H 6.0 were 46 mg·g^–1·h^–1 and 0.15 h^–1, respectively. In pH-regulated chemostat cultures, styrene was degraded at dilution rates ranging from 0.05 to 0.20 h^–1. Kinetic parameters and the proportion of each strain in the mixed culture were followed. At 0.20 h^–1, only one strain as compared to four initially present, remained in the medium. This strain Pseudomonas aeruginosa, degrades styrene with a specific activity of 293 mg·g^–1·h^–1. Such results could lead to industrial treatment of waste gas or water polluted with styrene. Correspondence to: J,-M. Lebeault