Biofilms isolated from washing machines from three continents and their tolerance to a standard detergent

The goal of this comparative study was to investigate biofilm forming microorganisms living in washing machines (WMs). Biofilms were sampled from 11 washing machines from four countries and three continents. Among the 94 isolated strains, 30% were potential human pathogens. Representative strains were selected and biofilm formation was evaluated with the crystal violet (CV) assay. The majority of the WM isolates formed more biofilm than their reference strains. Biofilms of P. putida WM (the largest biofilm producer) were exposed to different concentrations (0.0007–7 g l^?1) of the standard detergent IEC-A* at 30°C for 30 min and observed with confocal laser scanning microscopy. Using quantitative CVA, P. putida WM biofilm removal required higher detergent concentrations than the type strain. However, for both strains the recommended detergent concentration (7 g l^?1) was insufficient to completely clean surfaces from cell debris and exopolymeric substances.     

Biotransformation of isoeugenol to vanillin by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 cells

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.     

Effect of High Temperature on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> NBRI0987 Biofilm Formation and Expression of Stress Sigma Factor RpoS

Pseudomonas is an efficient plant growth–promoting rhizobacteria; however, among the limiting factors for its commercialization, tolerance for high temperature is the most critical one. After screening 2,500 Pseudomnas sp. strains, a high temperature tolerant–strain Pseudomonas putida NBRI0987 was isolated from the drought-exposed rhizosphere of chickpea (Cicer arietinum L. cv. Radhey), which was grown under rain-fed conditions. P. putida NBRI0987 tolerated a temperature of 40°C for ≤ 5 days. To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. P. putida NBRI0987 colony-forming unit (CFU)/ml on day 10 in both the absence and presence of MgSO₄.7H₂O (MgSO₄) in combination with glycerol at 40°C were 0.0 and 1.7 × 10¹¹, respectively. MgSO₄ plus glycerol also enhanced the ability of P. putida NBRI0987 to tolerate high temperatures by inducing its ability to form biofilm. However, production of alginate was not critical for biofilm formation. The present study demonstrates overexpression of stress sigma factor σ ^S (RpoS) when P. putida NBRI0987 is grown under high-temperature stress at 40°C compared with 30°C. We present evidence, albeit indirect, that the adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel regulatory process in which many different genes can be involved.     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Effects of circadian rhythms of fluctuating temperature on growth and biochemical composition of <Emphasis Type="Italic">Ulva pertusa</Emphasis>

The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20 ± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function of the thermal amplitude. Such a growth model can be described by G = β ₀ + β ₁(TA) + β ₂(TA)², where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β ₀ is the intercept on the G axis, and β ₁ and β ₂ are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at 20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga at the diel fluctuating temperatures in the present study. Handling editor: S. M. Thomaz     

Box-Behnken design in the development of optimized complex medium for phenol degradation using Pseudomonas putida (NICM 2174)

The biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different operating conditions. Box-Behnken design has been employed to study the effect of different experimental variables. Four variables of maltose (0.25, 0.5, 0.75?g/l), phosphate (3, 12.5, 22?g/l), pH (7, 8, 9) and temperature (30?°C, 32?°C, 34?°C) were used to identify the significant effects and interactions in the batch studies. A second order polynomial regression model, has been developed using the experimental data. It was found that the degrading potential of Pseudomonas putida (NICM 2174) was strongly affected by the variations in maltose, phosphate, pH and temperature. The experimental values were in good agreement with the predicted values, the correlation coefficient was found to be 0.9980. Optimum conditions of the variables for the growth of Pseudomonas putida (NICM2174) and for maximum biodegradation of phenol are maltose (0.052?g/l), phosphate (8.97?g/l), pH (7.9) and temperature (31.5?°C).     

Biosoftening of coir fiber using selected microorganisms

Coir fiber belongs to the group of hard structural fibers obtained from coconut husk. As lignin is the main constituent of coir responsible for its stiffness, microbes that selectively remove lignin without loss of appreciable amounts of cellulose are extremely attractive in biosoftening. Five isolated strains were compared with known strains of bacteria and fungi. The raw fiber treated with Pseudomonas putida and Phanerocheate chrysosporium produced better softened fiber at 30±2 °C and neutral pH. FeSO₄ and humic acid were found to be the best inducers for P. chrysosporium and P. putida, respectively, while sucrose and dextrose were the best C-sources for both. Biosoftening of unretted coir fibers was more advantageous than the retted fibers. Unlike the weak chemically softened fiber, microbial treatment produced soft, whiter fibers having better tensile strength and elongation (44.6–44.8%) properties. Scanning electron microscopy photos showed the mycelia penetrating the pores of the fiber, removing the tylose plug and degrading lignin.     

Arsenic in an As-contaminated abandoned mine was mobilized from fern-rhizobium to frond-bacteria via the ars gene

The mobilization of arsenic from fern-rhizobium to frond-bacteria in As-contaminated abandoned mine environments was found to be induced by the ars gene. Uncultured Acinetobacter sp. strain MRI67 (DQ539027) and uncultured Buttiaxella sp. strain MRI-65 (DQ539024) were identified in the root/rhizosphere based on the presence of the ars gene. The arsenite-oxidizing bacterial strains isolated in this study were found to grow in the presence of 14 mM sodium arsenite (NaAsO₂). In addition, the concentration of arsenic in five grams of Deparia lobatocrenata As-8 (EU476190) obtained from the Myoungbong abandoned mine area was 8,900 mg/kg ± 56.9, whereas the arsenic concentrations in the frond-rhizoplane of Pseudomonas putida GIST-MRP44-1 (EF623836), the root-rhizoplane of Acinetobacter sp. GIST-MRO62 (EF623849), and the stipe-rhizoplane of P. putida GIST-MRO63 (EF623850) were 41.9 mg/kg ± 1.0 (1 g of tissue), 32.9 mg/kg ± 0.9 (1 g of tissue), and 17.9 mg/kg ± 1.4 (1 g of tissue), respectively. Taken together, these findings suggest that the newly isolated indigenous fern-rhizobium, stipe-bacteria, and frond-bacteria may provide a better understanding of arsenic mobility in the field of molecular geomicrobiology and that it can be applied to the phytoremediation of arsenic-contaminated mines.     

Isolation and characterization of ethylbenzene degrading <Emphasis Type="Italic">Pseudomonas putida</Emphasis> E41

Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethyl-benzene as the sole carbon and energy source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μ_max) under the optimum conditions were 0.19+0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87+0.13 h⁻¹, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium (each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70–80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Survival of Heleomyza borealis (Diptera, Heleomyzidae) larvae down to − 60°C

Heleomyza borealis Boh. (Diptera, Heleomyzidae) overwinters as larvae in Arctic habitats, where they may experience winter temperatures below ? 15°C. The larvae freeze at c.? 7°C but in acclimation experiments 80% survived when exposed to ? 60°C. Of the larvae exposed to between ? 4 and ? 15°C, only 3% pupated. However, when cooled to ? 20°C this increased to 44%, with 4% emerging as adults. Larvae maintained at 5°C contained low levels of glycerol, sorbitol and trehalose, which did not increase with acclimation to low temperatures. However, levels of fructose increased from 6.1 μg mg^?1 fw in control animals to 17 μg mg^?1 fw when exposed to ? 2°C for 1 week. Larval body water (2.2 ± 0.1 g/g dw, mean ± SD, n = 100) and lipid content (0.22 ± 0.002 g/g fw, mean ± SE) showed no significant change during acclimation to low temperatures. Larvae maintained at a constant 5°C survived for over 18 months with little loss of body mass (from 7.5 ± 1.2 to 7.0 ± 1.2 mg fw, mean ± SD, n = 20), but none pupated. Heleomyza borealis larvae appear to feed and grow until they reach a body mass of about 7.5 mg and then become dormant. They remain in this state until they experience a low temperature stimulus (< ? 15°C) followed by a warm period (≈ 5°C). This ensures that the larvae pupate and adults emerge in early summer, allowing the maximum growing period before the following winter. Heleomyza borealis are adapted to survive long winters in a dormant larval state. They have a low metabolic rate, can conserve body water even at subzero temperatures but do not synthesize large quantities of cryoprotectants.     

Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Optimized prodigiosin production with Pseudomonas putida KT2440 using parallelized noninvasive online monitoring

The red pigment prodigiosin is of high pharmaceutical interest, due to its potential applications as an antitumor drug and antibiotic agent. As previously demonstrated, Pseudomonas putida KT2440 is a suitable host for prodigiosin production, as it exhibits high tolerance toward the antimicrobial properties of prodigiosin. So far, prodigiosin concentrations of up to 94 mg/L have been achieved in shake flask cultivations. For the characterization and optimization of the prodigiosin production process, the scattered light of P. putida and fluorescence of prodigiosin was measured. The excitation and emission wavelengths for prodigiosin measurement were analyzed by recording 2D fluorescence spectra. The strongest prodigiosin fluorescence was obtained at a wavelength combination of 535/560 nm. By reducing the temperature to 18 °C and using 16 g/L glucose, the prodigiosin concentration was more than doubled compared with the initial cultivation conditions. The obtained results demonstrate the capabilities of parallelized microscale cultivations combined with noninvasive online monitoring of fluorescence for rapid bioprocess development, using prodigiosin as a molecule of current biotechnological interest.     

Vanillin production using <Emphasis Type="Italic">Escherichia coli</Emphasis> cells over-expressing isoeugenol monooxygenase of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.     

Engineering Pseudomonas putida KT2440 for the production of isobutanol

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback‐resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y_Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical Y_Iso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non‐growing conditions.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition‐related alterations

Microorganisms, such as Pseudomonas putida, utilize specific physical properties of cellular membrane constituents, mainly glycerophospholipids, to (re‐)adjust the membrane barrier to environmental stresses. Building a basis for membrane composition/function studies, we inventoried the glycerophospholipids of different Pseudomonas and challenged membranes of growing cells with n‐butanol. Using a new high‐resolution liquid chromatography/mass spectrometry (LC/MS) method, 127 glycerophospholipid species [e.g. phosphatidylethanolamine PE(32:1)] with up to five fatty acid combinations were detected. The glycerophospholipid inventory consists of 305 distinct glycerophospholipids [e.g. PE(16:0/16:1)], thereof 14 lyso‐glycerophospholipids, revealing conserved compositions within the four investigated pseudomonads P. putida KT2440, DOT‐T1E, S12 and Pseudomonas sp. strain VLB120. Furthermore, we addressed the influence of environmental conditions on the glycerophospholipid composition of Pseudomonas via long‐time exposure to the sublethal n‐butanol concentration of 1% (v/v), focusing on: (i) relative amounts of glycerophospholipid species, (ii) glycerophospholipid head group composition, (iii) fatty acid chain length, (iv) degree of saturation and (v) cis/trans isomerization of unsaturated fatty acids. Observed alterations consist of changing head group compositions and for the solvent‐sensitive strain KT2440 diminished fatty acid saturation degrees. Minor changes in the glycerophospholipid composition of the solvent‐tolerant strains P. putida S12 and Pseudomonas sp. VLB120 suggest different strategies of the investigated Pseudomonas to maintain the barrier function of cellular membranes.     

Production of 3-hydroxypropionaldehyde using a two-step process with Lactobacillus reuteri

3-Hydroxypropionaldehyde (3-HPA) produced by Lactobacillus reuteri is a broad-spectrum antimicrobial substance of glycerol conversion. The aim of the present work was to optimize 3-HPA production by Lb. reuteri ATCC 53608 using a two-step process. The first step was the production of Lb. reuteri cells in optimal conditions. Cells were then harvested by centrifugation and suspended in glycerol solution, which the resting cells bioconverted to 3-HPA. The effect of biomass concentration, temperature, glycerol concentration, anaerobic/micro-aerophilic conditions, and incubation time was studied for high 3-HPA production. 3-HPA accumulation was limited by the death of cells in contact with high concentrations of 3-HPA. However, a very high 3-HPA concentration of 235±3 mM was obtained after 45 min of incubation at 30°C in 400 mM glycerol for an initial free-cell concentration of 1.6±0.3×10¹⁰ viable cells/ml. A high viability was maintained at low temperatures in the range 5–15°C, but with a slightly lower yield of 3-HPA at 5°C compared with higher temperatures, up to 37°C. Successive 1-h incubations of Lb. reuteri cells in 200 mM glycerol at 15°C to tentatively reuse the cells resulted in decreasing 3-HPA concentrations at the end of each cycle, with two successful production cycles yielding high 3-HPA concentrations of 147±1 mM and 128±2 mM.     

Oviposition pattern of the predator Podisus nigrispinus (Heteroptera: Pentatomidae) under different temperatures

The daily reproductive rate of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) fed with Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) larvae was studied at constant temperatures of 20, 23, 25, 28, 30 and 33±0.2°C, relative humidity of 60±10% and photoperiod of L:D 14:10. Daily reproductive rate of P. nigripinus was affected by age of this predator. Each P. nigrispinus female laid 5.3 (20°C) to 19.9 eggs/day (28°C) which developed into 4.3–16.5 nymphs, respectively. Highest daily reproductive rate of P. nigrispinus was recorded at 28 and 30°C for 5–30-day-old females. This predator showed higher daily reproductive rate than its prey A. argillacea at 25°C. It was also able to reproduce at temperatures from 20 to 33°C with maximum daily reproductive rate between 25 and 30°C. These results are important for optimizing mass rearing of P. nigrispinus in the laboratory.