Monitoring the acoustic activity of an aquatic insect population in relation to temperature,vegetation and noise

1. Acoustic population monitoring is a noninvasive method that can be deployed continuously over long periods of time and at large spatial scales. One of the newly discovered threats acting on biological diversity is anthropogenic noise. High levels of anthropogenic noise occur in aquatic environments, yet their effects on animals living in freshwater habitats have very rarely been investigated.
2. Here, we used acoustic monitoring and automatic detection to assess the acoustic activity of a population of a soniferous freshwater insect.
3. The sounds emitted by the corixid Micronecta scholtzi were recorded in a Mediterranean pond with an array of 12 hydrophones. An automatic analysis based on a measure of the amplitude found in the frequency band of M. scholtzi was developed to assess the level of acoustic activity. We used functional linear models, accounting for the periodicity of the calling behaviour, to estimate the possible effect of temperature, vegetation and a noise due to an immersed engine.
4. The automatic analysis was validated as an efficient method to measure the acoustic activity. The monitoring revealed a clear 24-hr pattern in the acoustic activity of M. scholtzi and three peaks of activity during the morning. Functional linear models revealed negative effects of both temperature and vegetation and showed that an engine noise, played back for 2 hr during the night, elicited an increase in the level of acoustic activity of the population. Moreover, a cross-correlation procedure showed that noise delayed the acoustic activity of the population.
5. Our results suggest that acoustic survey and automatic detection are efficient methods to monitor the acoustic activity of an insect population especially in response to an anthropogenic disturbance.

     

Automatic evaluation of antifungal volatile compounds on the basis of the dynamic growth process of a single hypha

An automatic analysing system was developed and employed for the evaluation of antifungal activity of volatile compounds in the gas phase. Aspergillus niger was inoculated on agar medium in the reaction vessel. The reaction vessel was incubated at 28° C for 24 h and then a volatile compound was introduced into the vessel either in a batch or flow manner. The antifungal activity of the respective compounds estimated in situ was expressed by the dynamic response parameters of a single hypha. All volatiles tested in the present system inhibited hyphal growth, except linalyl acetate: Limone and geraniol were the most inhibitory. In contrast, linalyl acetate promoted hyphal growth. By definition of the parameters, the fungicidal and fungistatic effects could be distinguished. Correspondence to: H. Matsuoka     

A high throughput profiling method for cutinolytic esterases

A procedure for identifying and profiling cutinolytic esterases was developed by combining traditional plate screen assays with an automated robotic system. In the first phase, the micro-organisms were screened on agar plates with cutin or the model substrate polycaprolactone as the sole carbon sources. In the second phase, p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The variables used were pH and the carbon chain length of the fatty acid moiety of the p-nitrophenyl substrate. Finally, ³H-labelled cutin was used as a specific substrate to verify the positive hits and to validate the screening procedure. With pH as the variable in the automatic screen, esterase production of cutinase positive strains typically proceeded in two stages: first an esterase with neutral activity optimum was produced, after which a strong esterolytic response in the alkaline range was detected. With carbon chain length of the fatty acid as the variable best correlation with cutinase production was obtained with strains showing a high ratio of activities towards p-nitrophenyl-butyrate and p-nitrophenyl-palmitate.     

Simultaneous on line monitoring of intracellular β-galactosidase activity and biomass using flow injection analysis inEscherichia coli batch fermentations

Summary A novel method to monitor on-line intracellular β-galactosidase activity and biomass simultaneously, using flow injection analysis (FIA), has been developed. The automatic ultrasonic cell disruption and FIA analysis allow the processing of 10 samples/hour with a wide and variable linear working range of β-galactosidase activity and biomass and a maximum relative standard deviation (RSD) of 1.5%. The system has been optimized by monitoring biomass and intracellular β-galactosidase activity inEscherichia coli batch fermentation.     

Measuring fig foraging frequency of the Yakushima macaque by using automatic cameras

An automatic camera system was employed to reveal the fig foraging frequency of primary seed dispersers on Yakushima Island, southern Japan. Seven automatic cameras were settled on sample branches of Ficus superba (Miq.) Miq. var. japonica Miq. to record animals foraging for figs. Figs on sample branches were counted at approximately 3 day intervals. The cameras took 168 photographs including 155 pictures of Yakushima macaques and two of birds, indicating that most of the figs at the inner parts of the crowns were eaten by the macaques. There was a linear relationship between the number of macaques foraging for figs on sample branches and the number of figs that dis-appeared within each period, suggesting that the automatic camera system was useful for estimating fig loss as a result of the foraging activity of the macaques on a branch basis.     

Esterase activity assay by flow injection analysis (FIA)

An automatic flow injection analysis (FIA) system for on-line determination of esterase activity has been developed. It is based on a colorimetric method using p-nitrophenyl propionate as substrate. The system permits a linear range analysis up to 0.18 U ml^–1, although the range can be extended up to 1 U ml^–1 without external dilution of the sample. The sampling frequency is of 4 samples per h with a relative standard deviation of 0.9%.     

Effective production of Pseudomonas fluorescens lipase by semi-batch culture with turbidity-dependent automatic feeding of both olive oil and iron ion

Summary An automatic feeding system to supply olive oil in semi-batch culture was established by monitoring cell concentration with a laser turbidimeter combined with a microcomputer and a pulse motor. In this automatic feeding system, specific olive oil supply rate (g olive oil) · (g dry cell)^-1 · h^-1, q ₀, was changed in an appropriate range. Attempts were made to produce lipase by a turbidity-dependent automatic fed-batch culture of Pseudomonas fluorescens. It was found from the semi-batch cultures with turbidity-dependent feeding of olive oil and with varied initial Fe ion concentrations that excess Fe ion was inhibitory to formation of the lipase. Turbidity-dependent automatic simultaneous feeding of olive oil and Fe ion was performed to obtain semi-deficiencies of both the oily substrate in the culture liquid and Fe content of the cells. Using this semi-batch culture, high lipase activity, 5600 units/ml, was attained at an optimal value of q ₀.     

Development of enzyme flow calorimeter system for monitoring of microbial glycerol conversion

Glycerokinase from Cellulomonas sp. was used to develop biosensor based on flow calorimetry for quantitative analysis of glycerol during bioconversion process. An automatic flow injection analysis device with the glycerol biosensor was built and tested during growth on glycerol of 1,3-propanediol-producing bacteria. The biosensor exhibited an extreme storage and operational stability enabling us to use it for more than 2 years without significant loss of sensitivity. No interference with 1,3-propanediol and fermentation medium was observed. The linear range of glycerol concentration up to 70 mM was extended by developed automatic dilution technique with the aim of automatic online monitoring of microbial process. The analytical system was able to monitor the bioconversion process in a fully automatic way during the whole run with sampling frequency of one sample per 10 min.     

A multiparameter phytoplankton culture system driven by microcomputer

A computer-controlled phytoplankton culture system is described which is regulated as a chemostat. Measurements of temperature, pH, size distribution, culture density,in vivo fluorescence, nitrate and nitrite, are automatic and programmable, thus obviating manual errors. The system has been developed successfully to survey long-term cultures of the dinoflagellateProrocentrum minimum. author for correspondence     

Analysis of glucose catabolites by using head-space gas chromatography for differentiation of some Gram-negative species

Volatile products from the degradation of glucose by a total of 66 strains ofEnterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, andPseudomonas aeruginosa were analyzed by head-space gas chromatography. The bacteria were incubated for 1 h in a glucose-containing buffer solution before being analyzed by gas chromatography, using manual, semiautomatic, and automatic head-space injection. From the chromatographic patterns obtained, all strains could be differentiated as to species, with the exception of two strains ofProteus mirabilis, which gave chromatograms similar to those produced byProteus vulgaris. The gas chromatographic head-space technique developed provides a rapid and easily performed means for identification of bacteria, particularly when using an automatic injection unit, as exemplified in this study on some of the Gram-negative species commonly encountered in urinary tract infections.     

Effect of hyperbaric stress on yeast morphology: study by automated image analysis

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO₂.     

Melanin Standard Method: Titrimetric Analysis

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48–53, 1992).     

Larval salt gland ofArtemia salina nauplii

Summary Naupliar brine shrimp (Artemia salina) have been used to study the salt-dependent regulation of protein synthesis. Measurement of thein vivo rates of protein synthesis was found to be very complex and dependent upon the leucine concentration of the external medium, rate of leucine entry and time of equilibration between various internal pools of leucine. Techniques were developed which permitted the measurement of rates of incorporation of^l4C-L-leucine into naupliar protein at various salinities under conditions that provided the organisms with a constant internal specific activity. It was found that salinities over 0.25 M NaC1 caused decreased rates of protein biosynthesis. A comparison of the rate of protein synthesis in the presence of chloramphenicol, cycloheximide and puromycin indicated that qualitative as well as quantitative changes in synthesis of proteins was directed by the external salinity. A feed-back mechanism based on the partitioning of available energy (ATP) between ion transport and protein synthesis is hypothesized.This work was supported by AEC grant RLO 2227-T13-1. The authors wish to express their gratitude to Prof. Robert R. Beoker and Robert L. Howard, Department of Biochemistry and Biophysics, for the technical assistance and use of the automatic amino acid analyzer in collecting data presented in Table 1 and Table 2.     

Nisin高产菌株的高通量筛选

【目的】乳酸链球菌素(nisin)是一种天然生物活性抗菌肽,对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,而用作食品的防腐剂。本研究通过建立高通量筛选方法,实现高效快速省力的高产菌株筛选,为工业上筛选高产菌株提供研究方案。【方法】通过对Lactococcus lactis ATCC11454菌株进行紫外诱变,获得2511株突变株。利用Biomek FXP自动工作站建立96微孔板的高通量筛选方法,突变株经高通量挑选、菌种培养及菌液稀释后,加入到生长至对数中期的藤黄微球菌中,采用改进后的比浊法快速检测nisin生物活性。用此方法对突变株进行初筛、复筛后可得到nisin高产菌株,并通过摇瓶发酵评估高通量筛选方法。【结果】确定比浊法检测的条件为:nisin活性稀释在10–25 IU/m L范围内,与藤黄微球菌反应2 h后检测藤黄微球菌的菌体量(OD600)。2511株突变株经过2轮高通量筛选,最终获得约50株产量提升的菌株,对其中8株进行摇瓶精确测量,显示产量均有提高,并且其中一株产量提升了30%,成功建立了高通量筛选nisin高产菌株的方法。【结论】利用比浊检测法,在其基础上成功建立高通量筛选高产nisin菌的方法,经过初筛复筛,整个周期由1人耗时5 d即可完成2511株突变株的筛选工作。相较于传统的选育方法,高通量筛选具有快速、稳定、高效的特点,提高了筛选效率,缩短了选育周期,是工业上筛选高产nisin菌的有效手段。     

Automatic laboratory-scale fed-batch procedure for production of recombinant proteins using inducible expression systems of Escherichia coli

Summary An automatic fed-batch procedure for the production of recombinant proteins in Escherichia coli was developed. Using glycerol as carbon source and by controlling the growth rate by using feed-forward algorithm, enabled high specific expression level (10–20 % of total cell protein) at high cell densities (20 g dry wt/l) to be achieved: rat and human soluble catechol-O-methyltransferase, calf prochymosin, and human troponin C were expressed with nearly 50-fold higher volumetric yield compared to the conventional (batch) procedures.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size,Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

Summary A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 x oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r=0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r=0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r=0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30–100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm× 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Measurement of α-amylase and glucoamylase activities produced during fermentation

A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α-d-glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.     

Evaluation of image analysis and laser granulometry for microbial cell sizing

A direct cell size measurement technique and an image analysis based sizing method were developed. The former consisted of a manual size measurement of the two-dimensional cell images on a video screen, with automatic data recording. This method was chosen as the reference. The latter, a semiautomatic method took advantage of a commercial computer program designed for image processing and particle morphology analysis. It gave average and median size values which were compatible with the manual method. However, the performance of these time consuming methods is limited. Hence, the laser granulometry technique, intrinsically far more powerful while capable of analysing millions of sample objects in a short time delay, was applied. The comparison revealed that this method gives too low size values, particularly in disagreement with the known dimensions of the bacterial (Zymomonas mobilis) cells. A size correction method was developed to realign the granulometry results ofZ. mobilis cell samples with those of the direct manual measurement method.     

Comparison of batch and semicontinuous cultures for production of protein from mesquite wood by Brevibacterium sp. JM98A

The production of protein by a Brevibacterium sp. JM98A usingmesquite wood as the substrate was compared in batch and semicontinuous cultures. A 14 liter glass fermentor with automatic pH, temperature, and foam control was used for the study. A pH range of 6.6 to 7.2 was optimum for the growth of JM98A. The batch and semicontinuous cultures were compared on the basis of viable cell counts, protein production, CMC-Ase (β-1,4-glucanase) activity, and filter paper cellulase (β-1,4-glucan cellobiohydrolyase) activity. Total hexose, cellulose, and reducing sugar consumption were measured. The semicontinuous process yielded 2.97 times as much protein in 72 hr as the batch cultures. Most of the biomass resulted from the utilization of soluble sugars rather than from the degradation of cellulose during the semicontinuous process.     

On-line determination of flocculating Saccharomyces cerevisiae concentration and growth rate using a capacitance probe

During continuous alcoholic fermentation, Saccharomyces cerevisiae 38A floc concentration was monitored using an on-line impedance probe. Since the sensor response is linear and does not depend significantly on yeast particle size, an automatic technique of determining the yeast growth rate has been developed and validated against the conventional mass balance method.