Application of mathematical optimization methods in microbiology

Mathematical methods of experiment design have so far found little use in the optimization of microbiological processes. The conventional optimization procedure is still the transformation of functional relationship ofn variables into n unidimensional optimizations; furthermore, the Bos-Wilson gradient method is often used. This paper presents a review of methods used in other fields, and their application in microbiological practice. The methods are classified according to whether they require, besides the simple determination of the objective function (direct search methods), also the finding of its first (gradient methods) or second derivative (Newton-Raphson method). A modified Rosenbrock’s method of direot optimum search and the gradient Box-Wilson method were used in parallel for the optimization of yeast growth on methanol. Their comparison showed that Bosenbrock’s method is more suitable for multiparameter systems.     

Plant species responses along a grazing disturbance gradient in Australian grassland

Abstract. Floristic data from paired roadside-paddock analyses from grassland in central Queensland, Australia, were ordinated. The mean direction of the vectors between these pairs was almost perfectly aligned with the indirect gradient represented by the first axis of Non-metric Multi-dimensional Scaling. It confirms anecdotal evidence of a trend from infrequently grazed roadsides to constantly grazed paddocks. The increasing abundance of annual herbs and grasses along this putative gradient is consistent with documented trends from elsewhere in the world. The response patterns of individual species along the disturbance gradient is consistent with ecological theory predicting unimodal peaks in abundance along physical environmental gradients. The ancestral perennial dominants of the grasslands, Dichanthium sericeum and D. queenslandicum, exhibited a declining response to grazing disturbance. Even the generally unpalatable perennial grass Aristida leptopoda declined considerably in the upper segments of the grazing disturbance gradient. A suite of herbaceous trailing legumes had peaks in their abundance near the middle of the grazing disturbance gradient, trends that can be readily explained by the combination of their palatability and intolerance to competition from tall perennial grasses. Several species including the noxious exotic herb Parthenium hysterophorus showed increasing abundance along the grazing disturbance gradient. The methodology may have application as a rapid method of assessing disturbance impacts elsewhere, and is most suited where a management differential between paired plots can be reliably generalized and where the physical environment is relatively monotonous.     

Multiple approaches to detect outliers in a genome scan for selection in ocellated lizards (Lacerta lepida) along an environmental gradient

Identification of loci with adaptive importance is a key step to understand the speciation process in natural populations, because those loci are responsible for phenotypic variation that affects fitness in different environments. We conducted an AFLP genome scan in populations of ocellated lizards (Lacerta lepida) to search for candidate loci influenced by selection along an environmental gradient in the Iberian Peninsula. This gradient is strongly influenced by climatic variables, and two subspecies can be recognized at the opposite extremes: L. lepida iberica in the northwest and L. lepida nevadensis in the southeast. Both subspecies show substantial morphological differences that may be involved in their local adaptation to the climatic extremes. To investigate how the use of a particular outlier detection method can influence the results, a frequentist method, DFDIST, and a Bayesian method, BayeScan, were used to search for outliers influenced by selection. Additionally, the spatial analysis method was used to test for associations of AFLP marker band frequencies with 54 climatic variables by logistic regression. Results obtained with each method highlight differences in their sensitivity. DFDIST and BayeScan detected a similar proportion of outliers (3–4%), but only a few loci were simultaneously detected by both methods. Several loci detected as outliers were also associated with temperature, insolation or precipitation according to spatial analysis method. These results are in accordance with reported data in the literature about morphological and life‐history variation of L. lepida subspecies along the environmental gradient.     

Isolation and characterization of CAC antenna proteins and photosystem I supercomplex from the cryptophytic alga Rhodomonas salina

In the present paper, we report an improved method combining sucrose density gradient with ion‐exchange chromatography for the isolation of pure chlorophyll a/c antenna proteins from the model cryptophytic alga Rhodomonas salina. Antennas were used for in vitro quenching experiments in the absence of xanthophylls, showing that protein aggregation is a plausible mechanism behind non‐photochemical quenching in R. salina. From sucrose gradient, it was also possible to purify a functional photosystem I supercomplex, which was in turn characterized by steady‐state and time‐resolved fluorescence spectroscopy. R. salina photosystem I showed a remarkably fast photochemical trapping rate, similar to what recently reported for other red clade algae such as Chromera velia and Phaeodactylum tricornutum. The method reported therefore may also be suitable for other still partially unexplored algae, such as cryptophytes.     

Habitat gradient and reproductive habits of the sevenStellaria species in Japan

The author studied the habitat preference, life form, life cycle and reproductive characteristics of the seven species of the genusStellaria along a valley in the northeastern part of Kyoto City. Along the valley 150 quadrats were set from the village up to the hills. The quadrats were sorted into 15 quadrat groups by their affinity in the environment. The quadrat groups were ordinated by the quotients of community and the Bray-Curtis method in order to reveal the complex gradient in the environment. The complex gradient could be best understood by the degree of human disturbance. The seven species showed a continuous distribution along the complex gradient. There existed three patterns in their habitat preference and reproductive characteristics: (1)S. media andS. neglecta grew as annuals in the habitat of severe human disturbance. (2)S. tomentella, S. sessiliflora, andS. diversiflora grew as perennials in the habitat of slight human disturbance. (3)S. aquatica andS. alsine grew as facultative annuals either in the habitat of severe or slight human disturbance.     

Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum

Chlorotetracyclin (10^-4M) has been used to observe the distribution of membrane-associated calcium during pollen germination of Lilium longiflorum. For comparison, the general membrane distribution has been determined with 4·10^-5 M fluorescamine. The pollen grains show a calcium gradient with either weak or strong chlorotetracycline-fluorescence intensity, but always increasing toward the germination colpus. This gradient intensifies during germination, reaching a maximum before the pollen tube emerges. The typical tip-to-base calcium gradient of the tube does not change during growth. Independent of the developmental stage, the pollen grains show a flat fluorescamine-fluorescence gradient with the highest intensity in one half of the grain. Pollen tubes reveal a tip-to-base membrane gradient, independent of their length. As an additional marker for membrane distribution, the distribution of phosphorus, measured by proton-induced X-ray emission in chemically fixed tubes, has been used. A tip-to-base phosphorus gradient, distinct from the calcium gradient measured with the same method, was detected.Abbreviation CTC chlorotetracycline     

目前最高分辨率的电泳──固相pH梯度等电聚焦

固相pH梯度等电聚焦是国际上80年代的新型电泳技术.利用一系列具有弱酸和弱碱性质的丙烯酰胺衍生物滴定时,在滴定终点附近形成的pH梯度并参与丙烯酰胺的共价聚合,从而形成固定的不随环境电场等条件变化的pH梯度,该方法具有比传统载体两性电解质等电聚焦更高的分辨率、更大的上样量.可用于分析和制备相近pI的蛋白质,多肽等.     

The effect of temperature and pH gradients on Lactobacillus rhamnosus gene expression of stress-related genes

In this study, Lactobacillus rhamnosus, a renowned probiotic, was cultivated in fluctuating environment. Base gradients caused by a pH control in an industrial process and temperature gradients caused by uneven heating were simulated with a scale-down method. A pH gradient was created in a plug flow reactor (PFR). Expression of pH stress-related genes (atpA, aldB, cfa, groEL, hrcA and pstS) were studied as a relative gene expression study using ldhD as a reference gene. Expression measurements were carried out with the TRAC method. The responses of groEL, hrcA and atpA genes to temperature and pH changes were observed. The expression of phosphate uptake system-related pstS gene was induced almost linearly in the chemostat cultivation experiments when the base gradient in the PFR was increased. Correlations between the results from gene expression studies and freeze stability or acid stress survival were studied. However, by measuring the expression of these genes, we were not able to predict eventual freeze stability or survival from the acid stress test.     

Phytosociological and environmental gradients in a sector of the Western Desert of Egypt

Summary The climatic and physiographic features of the Western Desert of Egypt vary gradually from the Mediterranean coast inland. The present study provides an analysis of the gradients exhibited by these environmental variations and their relationships with vegetational gradients and the phytosociological behaviour of species in a sector along the Cairo-Alexandria desert road. The multivariate analyses of the vegetation lead to the recognition of six interrelated vegetational groupings which were distributed along a climatic gradient of increasing aridity, and an edaphic gradient of increasing sandiness and decreasing salinity and level of total phosphorus. These groupings were distinguished into three sets: a set ofConvolvulus-Helianthemum-Artemisia, andHelianthemum-Echiochilon, a set ofAnabasis, andSalsola-Anabasis, and a set ofAsphodelus-Noaea andThymelaea-Anabasis-Noaea groupings. The first set occupies the more arid end of the climatic gradient and the end of the coarse texture, low salinity and low levels of total phosphorus of the edaphic gradient. The second set occupies the opposite end of the edaphic gradient and the middle position of the climatic gradient and the middle position of the climatic gradient, and the third, the middle position of the edaphic gradient and the less arid end of the climatic gradient. Taxonomic nomenclature is according to T?ckholm (1974). The multivariate analyses were carried out using a Burroughs B6700 at the Computer Center of Utah State University, and supported by the U.S. Desert Biome. The authors are indebted to W. Valentine, H. C. Romesburg and K. Marshall for their assistance with data processing.     

Adaptive latitudinal trends in the mandible shape of Apodemus wood mice

Aim Size and shape of the mandible are investigated across the latitudinal range of the European wood mouse (Apodemus sylvaticus), in order to address the relative importance of genetic structure, insularity, and geographical gradient in patterning morphological variation. Results are compared with those on two Asiatic species of wood mice, A. argenteus and A. speciosus. Location The European wood mouse is sampled by a set of trapping localities including both, islands and mainland populations, as well as the four genetic groups identified in previous studies. The localities cover a latitudinal gradient from 55° N to 36° N. Methods Different Fourier methods are applied to the outlines of mandibles and their results compared in the case of A. sylvaticus. All provide similar results and allow a quantification of the size and shape variations across the geographical range of the European wood mouse. Using the method allowing for the best reduction of the informative data set, a comparison of the European wood mouse with the two Asiatic species was performed. Results Within the European wood mouse A. sylvaticus, a strong latitudinal gradient in mandible shape overrides the influence of insularity and genetic structure. Yet, random morphological divergence in insular conditions can be identified as a secondary process of shape differentiation. Size displays no obvious pattern of variation, neither with insularity or latitude. A comparison with two other species of wood mice suggests that a similar latitudinal gradient in mandible shape exists in different species, mandibles being flatter in the north and wider in the south. Main conclusion The latitudinal gradient in mandible shape observed in the three species of wood mice is interpreted as an intraspecific adaptive response to gradual changes in feeding behaviour.     

Evaluation of methods for estimating soil carbon dioxide efflux across a gradient of forest disturbance

Better understanding of variation in soil carbon dioxide (CO₂) efflux caused by measurement techniques is needed, especially over gradients of site disturbance, to accurately estimate the global carbon cycle. We present soil CO₂ efflux data from a gradient of disturbance to ponderosa pine (Pinus ponderosa C. Lawson var. scopulorum Engelm.) forests in northern Arizona, USA that were obtained using four different techniques: vented static chambers, a Licor 6400‐09, and soil CO₂ diffusion profiles using two different models (Moldrup, Millington–Quirk) to estimate soil gas diffusivity. We also compared soil CO₂ efflux measured by the Moldrup and Millington–Quirk diffusion profile methods to nighttime total ecosystem respiration (TER) data from an eddy covariance tower. We addressed four questions: (1) Does the use of a given method to measure soil CO₂ efflux bias results across a disturbance gradient? (2) Does the magnitude of difference between observed and modeled estimates of soil CO₂ differ between methods and across sites? (3) What is the spatial variability of each method at each site? (4) Which method is closest to the estimate of TER measured by the eddy covariance tower? Although soil CO₂ efflux varied significantly among methods the differences were consistent among sites. Measured and modeled total growing season fluxes were generally higher for the Licor 6400‐09 and Millington–Quirk diffusion gradient methods compared with static chamber and the Moldrup diffusion gradient methods. A power analysis showed that the larger static chamber was the most efficient method at sampling spatial variation in soil CO₂ efflux. Nighttime measurements of soil CO₂ efflux from the Moldrup diffusion gradient method were most strongly related to nighttime TER assessed with eddy covariance. The use of a single, well‐implemented method to measure soil CO₂ efflux is unlikely to create bias in comparisons across a gradient of forest disturbance.     

Importance of the gradient in photosynthetically active radiation in a vegetation stand for leaf nitrogen allocation in two monocotyledons

Carex acutiformis and Brachypodium pinnatum were grown with a uniform distribution of photosynthetic photon flux density (PFD) with height, and in a vertical PFD gradient similar to the PFD gradient in a leaf canopy. Distribution of organic leaf N and light-saturated rates of photosynthesis were determined. These parameters were also determined on plants growing in a natural vegetation stand. The effect of a PFD gradient was compared with the effect of a leaf canopy. In Brachypodium, plants growing in a vegetation stand had increasing leaf N with plant height. However, distribution of leaf N was not influenced by the PFD gradient treatment. The gradient of leaf N in plants growing in a leaf canopy was not due to differences within the long, mostly erect, leaves but to differences between leaves. In Carex, however, the PFD gradient caused a clear increase of leaf N with height in individual leaves and thus also in plants. The leaf N gradient was similar to that of plants growing in a leaf canopy. Leaf N distribution was not affected by nutrient availability in Carex. In most cases, photosynthesis was positively related to leaf N. Hence, lightsaturated rates of photosynthesis increased towards the top of the plants growing in leaf canopies in both species and, in Carex, also in the PFD gradient, thus contributing to increased N use efficiency for photosynthesis of the whole plant. It is concluded that in Carex the PFD gradient is the main environmental signal for leaf N allocation in response to shading in a leaf canopy, but one or more other signals must be involved in Brachypodium.     

The Temperature Gradient Air–Soil as a Factor in the Optimization of Net Photosynthesis in Whole Plants

The effect of soil temperature on the net photosynthetic rate was studied by the method of multifactor analysis at early growth stages of narrow-leaved lupine (Lupinus angustifolius L.), white cabbage (Brassica capitata Lisg.), spring wheat (Triticum aestivum L.), cucumber (Cucumis sativus L.), tomato (Lycopersicon esculentum Mill.), and cotton (Gossypium hirsutum L.) plant species and cultivars contrasting in their heat demand. The optimum level of the net photosynthetic rate was observed in a wide range of soil and air temperatures, from cold- to heat-hardening temperatures, irrespective of the sign of the temperature gradient, whereas the magnitude and sign of the temperature gradient favorable for the highest potential net photosynthetic rate were species- and cultivar-specific and were not related to the cold tolerance of a species or cultivar.     

Determination of the G + C content of twoSyntrophus buswellii strains by ultracentrifugation techniques

An anaerobic syntrophic bacterial culture degrading benzoate was isolated from a river sediment. The syntrophic organism was grown in coculture in the presence of a hydrogenotrophic strain,Desulfovibrio fructosovorans orMethanospirillum hungatei. The G+C content of the syntrophic benzoate degrader determined by density gradient ultracentrifugation was similar to that ofSyntrophus buswellii (54.3%). A method ensuring the G+C% determination of syntrophic bacteria is presented.     

Isopycnic centrifugation of antigen-activated human blood lymphocytes in the early phase of in vitro stimulation

The interrelationship was determined between osmolarity, concentration, and density of aqueous solutions of polysucrose (Ficoll), metrizoate (Isopaque), polyethylene glycol, gum acacia, and ioglycaminic acid (Bilivistan). It was found that by mixing two solutions—each consisting of different concentrations of Ficoll, Isopaque, Tris-HCl buffer, and a Ringer solution—a linear density gradient could be produced, ranging from 1.055 to 1.095 g/ml. This gradient has a constant osmolarity of 280–290 mOsm (isotonic with human serum) and a constant pH of 7.4. A method is described for the determination of density distribution profiles of human blood lymphocytes by means of this gradient. The procedure has a high reproducibility and does not change the lymphocyte viability as determined by subsequent multiplication in culture and dye exclusion. The mean density of human blood lymphocytes, immediately after isolation, was found to be 1.072±0.0014 (n = 12). The yield of lymphocytes from the linear gradient is 50–70 %, but can be increased to 80–90 % if 1 % human albumin is included in the gradient material. From a discontinuous gradient 80–90 % of the cells may be recovered. Moreover, it was found that the same method may be used for the determination of the density profile of human spleen cells, thymus cells, granulocytes, thrombocytes, leukaemic lymphocytes, and lymphoid line cells.     

Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE)

The bacterial population during malolactic fermentation of Tempranillo wine was studied using the polymerase chain reaction-denaturing gradient gel electrophoresis, a culture-independent method successfully used for identification and monitoring of bacterial population in different habitats included food fermentations. The results showed that Oenococcus oeni was the predominant species in the malolactic fermentation of Tempranillo wines, although the presence of Gluconobacter oxydans, Asaia siamensis, Serratia sp., and Enterobacter sp. was also observed. These results were partly coincidental with those obtained from a culture-dependent method, using a selective medium. Therefore, it may be concluded that for a more complete knowledge of the bacterial community present during malolactic fermentation of Tempranillo wine, an approach that combines a culture-independent method and a culture-dependent method would be advisable.     

The selective isolation ofMicromonospora from soil by cesium chloride density gradient ultracentrifugation

Summary A novel method is described for the selective isolation ofMicromonospora from mixed microbial populations in soil. Microorganisms were released from soil by sonication, and the bulk of the soil debris was discarded by low-speed centrifugation. The supernatant microbial suspension was concentrated and applied to a continuous, linear (1.1–1.6 g/ml) gradient of CsCl which was then centrifuged at high speed. The gradient was fractionated, and each fraction was diluted and plated on a medium devoid of antimicrobial agents.Micromonospora were found in the 1.35–1.42 g/ml density band. Occasionally,Bacillus species were also obtained in this density range, but other nonfilamentous bacteria or actinomycetes were usually not observed. This technique allowed the isolation of portions of the soilMicromonospora population which were suppressed by conventional isolation techniques employing heat and antibiotics.     

High Resolution Polyacrylamide Gradient Gel Electrophoresis

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e. g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%)and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickett-sii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.     

Genomic scanning using AFLP to detect loci under selection in the moss Funaria hygrometrica along a climate gradient in the Sierra Nevada Mountains,Spain

The common cord moss Funaria hygrometrica has a worldwide distribution and thrives in a wide variety of environments. Here, we studied the genetic diversity in F. hygrometrica along an abiotic gradient in the Mediterranean high mountain of Sierra Nevada (Spain) using a genome scan method. Eighty‐four samples from 17 locations from 24 to 2700 m were fingerprinted based on their amplified fragment length polymorphism (AFLP) banding pattern. Using PCA and Bayesian inference we found that the genetic diversity was structured in three or four clusters, respectively. Using a genome scan method we identified 13 outlier loci, which showed a signature of positive selection. Partial Mantel tests were performed between the Euclidean distance matrices of geographic and climatic variables, versus the pair‐wise genetic distance of the AFLP dataset and AFLP‐positive outliers dataset. AFLP‐positive outlier data were significantly correlated with the gradient of the climatic variables, suggesting adaptive variation among populations of F. hygrometrica along the Sierra Nevada Mountains. We highlight the additional analyses necessary to identify the nature of these loci, and their biological role in the adaptation process.     

An Improved Method for the Isolation of Highly Polymerized Native Deoxyribonucleic Acid from Certain Protozoa*

A relatively simple phenol extraction method, with EDTA as the nuclease inhibitor, is described for the isolation of purified, highly polymerized native DNA from Trichomonas vaginalis, Trichomonas gallinae, and Tritrichomonas foetus; it is applicable also to Tetrahymena pyriformis. RNase Tl, RNase A (Worthington's R), pronase, and α-amylase digestions constitute important steps in obtaining satisfactory yields of DNA. High degree of polymerization of the isolation product was estimated by hyperchromicity at O.D.₂₆₀ after DNase treatment and by CsCl gradient analysis. The double-stranded condition of the DNA samples was estimated by the latter method and by denaturation with NaOH, and the molecular weight by sucrose gradient analysis. Purity of the samples was determined spectrophotometrically and by chemical analyses for protein and glycogen. DNA percent recovery was estimated by the diphenylamine reaction.