William E. Inniss

Measurement of Electron Transport System (ETS) activity in soil.

Electron transport system (ETS) activity was measured in amended and nonamended soil by measuring the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan), which can be easily extracted with methanol without interference from other compounds found in soil. A high correlation between ETS activity and oxygen consumption was observed. This technique allows rapid quantitative measurements of microbial ETS activity in soil.

PHENOL DEGRADATION BY A PSYCHROTROPHIC STRAIN OF PSEUDOMONAS PUTIDA

SummaryCell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, So, ranging from 14 to 1000 mg/1. The experimental data for 14<=So<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: μm=0.119 h−1, KS=5.27 mg/1 and KI=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.

Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature.

Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax. The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively. For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megaterium and B. psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

Effect of ph on the methylation of mercury and arsenic by sediment microorganisms

Abstract The effect of pH on the methylation of mercury and arsenic in lake sediment amended with nutrients was examined. The formation of methyl mercury from inorganic mercuric chloride occurred only in the pH range of 5.5–6.5. Dimethyl mercury was not produced. Mercury methylation in this narrow pH range was probably the result of a microbial population which had adapted to the in situ pH of 5.8. The levels of methyl mercury were reduced in the presence of sulfuric acid, probably due to sulfide formation. In the arsenic methylation experiments, volatile arsine or methyl arsines were rarely detected. Analysis of the culture fluid revealed that methyl arsonic acid and dimethyl arsinlcacid were formed over the pH range of 3.5 to 7.5. In contrast to the mercury methylation experiments, the arsenic methylating microorganisms were not as sensitive to changes in pH and different microorganisms may have been responsible for methylating arsenic over this pH range.

Impact of volatile aromatic hydrocarbons, alone and in combination, on growth of the freshwater alga Selenastrum capricornutum

Abstract A sealed, air-tight exposure system was employed for toxicity testing of volatile aromatic hydrocarbons. Within the sealed flasks, growth of the representative green alga, Selenastrum capricornutum , was inhibited due to the restriction of gas exchange, but inhibition was overcome by the addition of NaHCO 3 (0.4% w/v) to the growth medium. Toxicity was determined as the concentration required to reduce growth by 50% following an 8-day exposure period (EC 50 ). Relative toxicity of 6 aromatic hydrocarbons was, in order of increasing toxicity, benzene (EC 50 =41.0 mg/l), toluene (9.4 mg/l), ethyl-benzene (4.8 mg/l) and p -, m -, o -xylene (between 3.9–4.4 mg/l). Examination of the toxicity of hydrocarbon mixtures revealed an additive toxicological interaction between benzene, toluene and m -xylene, indicating that the toxicity of a mixture can be predicted from the sum of the toxicities of each component. A synergistic interaction was evident between benzene and toluene at higher concentration levels, but the interpretation of synergism is complicated by a rapid increase in sensitivity of algae to greater toluene concentrations.

THE RELATIONSHIP BETWEEN TOXICITY AND BIOCONCENTRATION OF VOLATILE AROMATIC HYDROCARBONS BY THE ALGA SELENASTRUM CAPRICORNUTUM

Abstract Bioconcentration potentials determined for benzene, toluene, ethylbenzene and 3 xylene isomers (para, meta and ortho) using the alga Selenastrum capricornutum were found to range from 1.63 [log=μg/kg/μg/L] for benzene to 2.41 for p -xylene. A strong linear correlation was evident between bioconcentration and the octanol/water partition coefficient (Kow), as well as between bioconcentration and toxicity. Two intracellular components (cell wall and microsome) were isolated and examined for their ability to bioconcentrate aromatic hydrocarbons. The microsome component was found to bioconcentrate aromatic hydrocarbons to the same degree as the whole cell, while the cell wall component showed a more limited capacity for bioconcentration.

Perturbation of lipid membranes by organic pollutants

The ability of a range of organic pollutants-hexachlorobenzene, mirex(1,1a, 2,2,3,3a,4,5,5,5a,5b,6-dodecachlorooctahydro-1,3,4-metheno-1H cyclobuta(cd) pentalene), 1,3,5-trichlorobenzene, 2,4,6-trichlorophenol,p-nitrophenol,p-chlorophenol, DDT, and pentachlorophenol to perturb liposomes of dipalmitoyl phosphatidylcholine (DPPC) has been measured by differential scanning calorimetry. The degree of perturbation was measured by the increase in breadth of the main DPPC phase transition in both heating and cooling scans. DDT and the phenol derivatives were effective perturbers of phospholipid, broadening the transition by as much as 12-fold. Hexachlorobenzene and mirex did not perturb at all when mixed with DPPC at concentrations as high as 20 mol%, although 1,3,5-trichlorobenzene caused slight broadening of the main transition at this concentration. Perturbation is facilitated by the presence of a hydroxyl group on the benzene ring and hindered by increasing degrees of chloride substitution. An apparent correlation exists between the extent of phospholipid perturbation measured by differential scanning calorimetry and LD50 values for these compounds taken from the literature. This suggests the possibility of formulating an “index of perturbation” which could be used to screen certain classes of organic compounds for potential biological toxicity on a routine basis.

Thermotolerance, cell filamentation, and induced protein synthesis in psychrophilic and psychrotrophic bacteria

Both the psychrophile Aquaspirillum arcticum and the psychrotroph Bacillus psychrophilus were found to acquire thermotolerance when either heat shocked or treated with nalidixic acid; two conditions which also resulted in the induction of heat shock proteins and/or stress proteins and also cell filamentation. The possible relatedness of acquisition of thermotolerance and cell filamentation was examined by inhibiting cell filamentation with 1.5% KCl. A. arcticum cells which were heat shocked in the presence of KCl did not become filamentous nor acquire thermotolerance suggesting that these two responses may be related. On the other hand, when cells of B. psychrophilus were treated in a similar fashion, they also were prevented from cell filamentation but their ability to become thermotolerant was unaffected. When A. arcticum cells were heat shocked in the presence of chloramphenicol, heat shock protein synthesis was inhibited but not the acquistion of thermotolerance. Similar experiments with B. psychrophilus revealed that partial induction of heat shock proteins still occurred; however, no thermotolerance was exhibited.

Detection and analysis of interactions between atrazine and sodium pentachlorophenate with single and multiple algal-bacterial populations.

A multispecies-multitoxicant defined microcosm was studied by examination of its various components. Determination of population interactions showed thatChlorella vulgaris inhibitedAnkistrodesmus braunii by 32%. The presence ofChromobacterium violaceum resulted in a further inhibition ofA. braunii (17%) but its presence had no effect onC. vulgaris. Sensitivity of the two algae and the bacterium were determined for the toxicants atrazine1 and sodium pentachlorophenate, both singly and in combination. While both algae were sensitive to the toxicants, the combined effect of the toxicants on the individual populations was additive. In mixed cultures with one toxicant present, a synergistic interaction was detected between sodium pentachlorophenate andC. vulgaris with respect to A.braunii. Further, the combination ofA. braunii and atrazine withC. vulgaris resulted in an increased inhibition ofC. vulgaris. These observations indicate that biological-chemical interactions can occur as do population-population and chemical-chemical interactions. When mixed cultures of the algae were treated with both toxicants, chemical antagonism was observed. This antagonism was modified by the presence of the bacterium but it was not eliminated. It was concluded that the techniques allowed determination of the interactions in a complex microcosm.

Development of an optimal heterotrophic growth medium for Chlorella vulgaris

SummaryFinal biomass yields of Chlorellavulgaris cultured heterotrophically in bristol medium amended with 0.1% (w/v) yeast extract (Difco) or 0.5% glucose (w/v) were 26 and 58 times higher, respectively, than yields obtained for autotrophically grown cells in the light. Similarly, final biomass increases were 35 and 138 fold for these organic substrates in the dark. The mixture of 0.1% yeast extract and 0.5% glucose was optimal and produced increases in final biomass of 70 and 140 times in the light and dark, respectively.