Use Of Organisms Research Articles

Live cultures: Their use in industrial biotechnology

Advances in the areas of molecular genetics and molecular biology provide a tremendous opportunity for development of custom-made microorgansims for human and animal use. This reviews discusses the current and future potentials of these organisms for use in the food and agricultural industries.

Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains

Vibrio cholerae may be a particularly effective organism for use in delivering heterologous antigens to stimulate a common mucosal immune response. A live attenuated vaccine strain of V. cholerae was constructed from the ctxA deletion mutant 0395-N1, containing the B subunit of Shiga-like toxin I under the transcriptional control of the iron-regulated irgA promoter. The B subunit of Shiga-like toxin I is identical to the B subunit of Shiga toxin (StxB). irgA encodes the major iron-regulated outer membrane protein of V. cholerae, which is a known virulence factor for this organism. Clones of the structural gene irgA from the classical V. cholerae strain 0395, with the gene for the Shiga-like toxin I B subunit inserted under the control of the irgA promoter, were used to introduce an internal deletion of irgA into the chromosome of 0395-N1 by in vivo marker exchange, using the suicide vector plasmid pCVD442. This plasmid contains the sacB gene from Bacillus subtilis, which allowed positive selection for loss of plasmid sequences on exposure to sucrose. The construction of vaccine strains was confirmed by Southern hybridization studies and outer membrane protein analysis. The expression of StxB in the vaccine strain VAC2 following growth in high- or low-iron conditions was shown to be tightly iron-regulated by Western blot analysis and by quantification of StxB using a sandwich enzyme-linked immunosorbent assay. The production of StxB by VAC2 under low-iron conditions was greater than that of the reference strain Shigella dysenteriae 60R.(ABSTRACT TRUNCATED AT 250 WORDS)

Use ofNeomysis mercedis(Crustacea: Mysidacea) for Estuarine Toxicity Tests

The mysid Neomysis mercedis was examined as a test organism for use in acute toxicity tests at intermediate salinities characteristic of estuarine waters. Several sensitive invertebrate species are available for marine assessments (mysids) and freshwater tests (cladocerans), but few are available for estuarine toxicity tests. Observations in the laboratory indicate that Neomysis mercedis can be reared successfully at a temperature of 17°C, a salinity of 2‰, and a population density less than 5/L. Brine shrimp nauplii Artemia salina, algae, and commercial foods were used to sustain mysid cultures. Neomysis mercedis is viviparous and can complete its life cycle in 3–4 months. Neomysis mercedis is as sensitive as or more sensitive to toxicants than the marine mysid Mysidopsis bahia and the freshwater cladocerans Daphnia magna, Ceriodaphnia dubia, and Simocephalus serrulatus. The mean 96-h LC50 values (concentrations lethal to half the test animals) for N. mercedis, in increasing order, were 0.20 μg/L for methyl parathion, 2.2 μg/L for malathion, 14 μg/L for carbofuran, 150 μg/L for copper sulfate, 280 μg/L for thiobencarb, and 1,600 μg/L for molinate. Neonates (5 d postrelease) were generally more sensitive than older juveniles. Coefficients of variation (100·SD/mean) of LC50 values varied from 21 to 35%.

Direct microbial conversion

Process development is reviewed for ethanol production from cellulosic biomass via direct microbial conversion (DMC). Experimental data addressing cellulase production and ethanol tolerance are also presented for the candidate DMC organismsClostridium thermocellum andClostridium thermosaccharolyticum. Two potential paths are identified for obtaining organisms for use in DMC. Path 1 involves modification of excellent ethanol producers, so that they also become good cellulase producers; Path 2 involves modification of excellent cellulase producers, so that they also become good ethanol producers. Cellulase production, ethanol tolerance, and ethanol selectivity are considered for both Path 1 and Path 2 organisms. It is concluded thatin situ cellulase production has the potential to allow cost reductions relative to state-of-the-art process designs on the order of 50¢/gal. Based on the data available, the value of cellulase production bythermocellum corresponds to 90% of this amount. However, each process path has a strategic obstacle to be overcome: high-level cellulase expression and secretion for Path 1, and high ethanol selectivity for Path 2. Ethanol tolerance is not seen as a primary factor in choosing between DMC and other ethanol process alternatives.

Predicting the toxicity of bulk sediments to aquatic organisms with aqueous test fractions: Pore water vs. elutriate

The objective of this research was to evaluate the acute toxicity of bulk sediment vs. pore water or elutriate to fathead minnows (Pimephales promelas), cladocerans (Ceriodaphnia dubia), amphipods (Hyalella azteca), and oligochaetes (Lumbriculus variegatus). A total of 29 different samples from seven sites were used for this analysis. In most instances, pore water was more toxic than corresponding elutriate. Based on the results of these assays, pore water, but not elutriate, was an effective test fraction for predicting the presence of bulk sediment toxicity; however, there were a number of observations of pore water toxicity in the absence of bulk sediment toxicity. Data from this study concerning the sensitivity of Pimephales promelas, Hyalella azteca, and Lumbriculus variegatus to bulk sediment vs. pore water or elutriate suggest that upper-water-column species are inappropriate organisms for use in bulk sediment tests focused on evaluating in situ toxicity to benthic species. Hyalella azteca was the most sensitive and Lumbriculus variegatus the least sensitive species evaluated.

Reptilian embryos: A model for sex determination and sexual differentiation in amniotes

Researchers realize that the selection of organisms for use as models requires both practical (e.g., abundance, availability, ease of care, and size) and scientific (appropriateness of the model) considerations. Based upon both of these considerations the use of embryonic reptiles as models for studies focusing on sex determination and sexual differentiation in amniotes is suggested. Phylogenetically, the class Reptilia gave rise both to birds and mammals, and ontogenic-ally, reptiles are very similar to these groups. Among amniote vertebrates, reptiles are unique in that they exhibit two distinct kinds of sex-determining mechanisms: genotypic sex determination (GSD) and temperature-dependent sex determination (TSD). In addition representatives of this group exhibit diverse patterns of sex chromosome morphology ranging from male heteromorphic to homomorphic, to female heteromorphic. This diversity has provided researchers with unique opportunities to address questions regarding the physiology of gonadal determination. This paper reviews recent studies which have demonstrated that 1) GSD and TSD may have a similar molecular basis; 2) steroid hormones can override the normal sex-determining mechanism both in species with TSD and GSD; 3) the embryonic period of hormone sensitivity coincides with the period of normal gonadal differentiation; and 4) once a gonad has differentiated it is apparently committed and cannot be “sex-reversed.” In addition, conflicting reports regarding the effects of exogenous hormones on species exhibiting TSD and those with heteromorphic sex chromosomes are considered from the point of view that such variations are the result of differences in mechanisms controlling the various levels of sexuality.

Theoretical Issues in the Regulation of Genetically Engineered Organisms: The Case of Deliberate Release

At the cutting edge of research in biotechnology is the production of special genetically engineered organisms for use in an open, agricultural environment. We argue that the standard approaches to risk assessment and management employed by regulatory agencies are inadequate to the task of properly assessing the advisability of such experiments, as well as the even more significant prospect of widespread commercial use of genetically engineered organisms in agriculture. Given the current lack of sufficient information with which to make competent assessments of the risks involved and the attempts to gain such information in a timely fashion, we conclude that a compelling case for deliberate release programs has not been made.

Resistance to Insecticides in the Common Green Lacewing Chrysoperla carnea (Neuroptera: Chrysopidae) in Southern Ontario

Populations of Chrysoperla carnea (Stephens) were resistant to a wide range of insecticides, including most of the commonly used groups (pyrethroids, organophosphorous insecticides, carbamates) used in orchards. Resistance was partially because of enhanced metabolism of pesticides by resistant C. carnea larvae. Metabolism was by oxidative and esteratic routes; the relative importance of the two systems depended upon the pesticide. In laboratory cultures, resistance to organophosphorous insecticides declined approximately 10 times (3 times for pyrethroids) over 3 yr, suggesting that careful quality controls are required for mass-rearing programs. Provided that resistance can be stabilized at high levels in the laboratory colony, the wide range of resistance shown here makes C. carnea a prime candidate for mass releases of pesticide-resistant beneficial organisms for use in IPM programs.

Freshwater Sediment Toxicity Bioassessment: Rationale for Species Selection and Test Design

The rationale and conceptual basis for the use of sediment toxicity assays are discussed in relationship to their use in sediment evaluations employing faunal surveys, toxicity assays, and chemical analyses. The disadvantages and advantages of various species from the major classes of aquatic organisms for use as sediment toxicity assay organisms are presented. Relative sensitivities of selected species and their ease of laboratory culture and utility as assay organisms are used to rank assays and propose a battery of assays for the rapid screening evaluation of sediment toxicity. The usefulness of a battery of assays for the screening evaluation of sediment toxicity and statistical considerations which are important in the development of study designs and the analysis of results from the battery of proposed assays are discussed. The assays recommended for inclusion in the screening battery for evaluation of sediment toxicity are Microtox ®, an algal assay, the Chironomus tentans 10-d growth assay, and the 48-h Daphnia magna acute assay.

Radiation survival of two nalidixic acid resistant strains of Salmonella typhimurium in various media

Radiation doses required for 90% inactivation, the D 10 values, have been determined for two nalidixic acid-resistant strains of Salmonella typhimurium, Nal R ATCC 13311 and K1-2B, in different media. The D 10 values were 0.20, 0.57 and 0.53 kGy for the ATCC 13311 strain, and were 0.21, 0.4 and 0.32 kGy for the K1-2B strain, in phosphate buffer, in nutrient broth and on chicken drumsticks, respectively. Since these two strains have radiation sensitivity similar to several Salmonella serotypes reported in the literature, they are good indicator organisms for use in studies on the effect of irradiation on Salmonella in foods that might frequently be contaminated with such organisms.

Fermentation Characteristics of Lactobacilli in Okra (Hibiscus esculentus) Juice

ABSTRACTOkra juice was fermented with nine strains of homofermentative and ten strains of heterofermentative lactobacilli as well as one commercial culture to study the metabolic activities of these cultures in an effort to select organisms for use in okra fermentation. During a 24 hr fermentation one Lactobacillus cellobiosus strain converted 29.7% of the mucilage, utilized 85.7% of the reducing sugars and 90.3% of the sucrose decreasing the pH value to 3.65. During the same period seven other strains converted from 20.2–26.8% of the mucilage, 78–98% of the reducing sugars and 75–80% of the sucrose giving a pH value between 3.45 and 4.23. All strains showed considerable variation in the ability to produce lactic acid, acetic acid and ethanol, and they all possessed low levels of lactate dehydrogenase activity.

EXPERIMENTAL SYSTEMATICS—LA DUKE'S REFRESHING COUNTERPOINT OF VIEW

John La Duke (1987) brings up many worthwhile points in his counterpoint of view of Gilmartin (1986). The crux of my paper, however was to support a concept sensu lato of experimental research, i.e., one dependent upon the essential element of hypothesis-testing rather than upon manipulation of organisms as implied in Hagen (1983). Whether or not a given research paper deals with hypothesis-testing implicitly or explicitly is another interesting question. La Duke has done us a favor also by cogently emphasizing the importance of carefully distinguishing: (1) the heuristic element of research (hypothesis-generation) from (2) hypothesis-testing. Both of these may occur and usually do occur in such rapid succession that they are difficult to distinguish but as La Duke points out, experiments need to be planned that explicitly attempt to falsify the hypothesis. Certainly, it is true that the latter effort is NOT evident in the majority of plant systematics papers, and I might add, it need not be so in every paper. Nonetheless, the more plant systematic research that is carried out with the world view of La Duke and others whom he cites, the richer will be the area of plant systematics, an already highly rewarding subdiscipline.

Methods for making kefir and fermented milks based on kefir

SUMMARYFlavour, aroma and appearance of milks fermented with commercially available kefir grains were compared with similar characteristics of heat treated (90 ΰC/30 min) milks that had been fermented with aroma producing starters in addition to the kefir grains. The starters used were either a mixture ofStreptococcus lactissubsp.diacetilactisandLeuconostoc cremoris(lactic culture) or ofStr. thermophilusandLactobacillus bulgaricus(yogurt culture).Two methods of fermentation were investigated involving removal of the grains followed by (i) a second fermentation of the kefir with the aroma-producing organisms and (ii) combining the kefir with aroma-producing organisms for use as a starter to ferment a further sample of heat-treated milk. Products were assessed by a taste panel and marked for acceptability. Addition of lactic or yogurt cultures resulted in products with increased mean scores for aroma and more acceptable flavours.

Adherence of Lactobacillus Species to Human Fetal Intestinal Cells

Thirty-two lactobacilli were tested for ability to adhere to a human fetal intestinal epithelial cell line. By an in vitro system, two adherence mechanisms were found. One mechanism, requiring calcium in the adherence reaction, was nonspecific and allowed all lactobacilli tested to adhere. The other system, not requiring calcium, was found in four strains, all human Lactobacillus acidophilus isolates. Colonial morphology, serial broth passage, and exposure of cell crops to freezing or lyophilization did not affect adherence of Lactobacillus acidophilus. In vitro adherence, combined with subsequent in vivo studies, may provide a basis for screening candidate organisms for use in microbiotic supplements.

Fungal metabolism of (−)-deprenyl and pargyline

Deprenyl and pargyline were readily metabolized by Cunninghamella echinulata ATCC 9244 and the products obtained were identified by gas-liquid chromatography and mass spectrometry. Deprenyl was completely metabolized to four products; amphetamine, N-methylamphetamine, 1-phenyl 2-propanone oxime and N-acetylamphetamine. Pargyline metabolism extracts contained substrate and five products; N-propargylbenzylamine, N-hydroxy-N-propargylbenzylamine, N-methylbenzylamine, N-acetylbenzylamine and benzylamine. Substrate concentration influenced the relative amounts of metabolites recovered. C. echinulata is an excellent organism for use as a model of mammalian metabolism.

The rock oyster Saccostrea glomerata as an indicator of trace metals in Hong Kong

An urgent need for a sub-tropical and tropical indicator organism for use in the biological monitoring of pollutants is recognised. To this end, preliminary studies have been undertaken to test the indicator ability of the rock oyster Saccostrea glomerata (=Crassostrea glomerata Gould) for monitoring the trace metals cadmium, copper, iron and zinc. A survey of these trace metals in rock oysters from 54 sites in Hong Kong waters revealed elevated levels of metals in several areas. Profiles of metal abundance in S. glomerata agreed substantially with the distribution of industrial and domestic discharges in Hong Kong, and also with patterns of contamination found in studies of the Pacific oyster Crassostrea gigas and of sediments. Application of POLYDIV statistical analysis to these data has aided their interpretation and confirmed the relationship between known areas of contamination and elevated metal levels in S. glomerata. This oyster, which exhibits an unusually great capacity to accumulate a range of elements from the ambient environment, thus appears to be a reliable indicator organism, and may be of great value in global monitoring studies.

Lichen transplants as biological indicators of SO2 air pollution in Copenhagen.

It is concluded from the high correlation between visible transplant damage and SO2 levels, that SO2 pollution gradients can be sufficiently described by means of the lichen transplantation method. The transplanted lichen species should be chosenon the basis of its SO2 sensitivity, this being neither too high nor too low in relation to the average SO2 level of the area under investigation. A description of the existing lichen vegetation or a knowledge of the SO2 variation in the area should thus precede any transplantation experiments.Hy-pogymnia physodes, which was also used successfully in transplantation experiments in Oslo (KROG & BRANDTH, 1975), showed sufficient variation in degree of damage after 6 months of transplantation in the Copenhagen area to be a useful indicator of SO2 air pollution levels. Thus, our data suggest that Hypogymnia physodes is a suitable organism for use in future transplantation experiments on air pollution levels in metropolitan areas in southern Scandinavia.

Phenylalanine tolerance tests in simian primates.

The metabolism of amino acids by nonhuman mammalian organisms is of interest in evaluating such organisms for use as animal models of human amino acid metabolic disorders (1). Of particular interest in this regard is phenylketonuria, a genetically-determined deficiency of liver phenylalanine hydroxylase activity that results in impaired metabolism of phenylalanine and has only been discovered in humans. One approach to the production of an animal model for phenylketonuria has been the feeding of large amounts of phenylalanine to rats and monkeys (2, 3). While this reproduces certain biochemical and clinical abnormalities also noted in phenylketonuria, many other characteristics, including the high tyrosine concentrations and the significant phenylalanine hydroxylase activity (3, 4) are distinctly different from human phenylketonuria. Two strains of mice have been reported to have deficient metabolism of phenylalanine (5, 6). However, the “dilute-lethal”strain (so named from its dilute hair color and early death of homozygotes) has recently been shown to metabolize phenylalanine indistinguishably from other mouse genotypes when on a normal diet (7); and in the “wabbler-lethal”strain (with defects in gait and lethality in homozygotes) the plasma phenylalanine concentrations and the plasma phenylalanine: tyrosine ratios are much lower than in human phenylketonuria. In addition the liver phenylalanine hydroxylase activity in this latter strain is much higher in relation to controls than is the case in human phenylketonuria (6). Consequently, there is still a need to develop a genetically-determined animal model for phenylketonuria. One approach to the production of inherited phenylketonuria in animals is the purposeful mating of known heterozygotes for this disorder. Certain studies in humans have indicated that intravenous phenylalanine tolerance tests are useful in differentiating heterozygotes for phenylketonuria from normal individuals (8-10). For several years, therefore, we have employed intravenous phenylalanine tolerance tests as a technique for identifying primate carriers of the mutant gene for phenylketonuria for the purposes of mating these animals.

Selection of test organisms for use in evaluating microbial inhibitors in fuel-water systems.

Selection of test organisms for use in evaluating microbial inhibitors in fuel-water systems.

Selection of test organisms for use in evaluating microbial inhibitors in fuel-water systems.

Selection of test organisms for use in evaluating microbial inhibitors in fuel-water systems.