Endotoxin inactivation in water by using medium-pressure UV lamps.

Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be approximately 0.55 endotoxin unit/ml per mJ/cm(2) of irradiation delivered.

Transfer and expression of mesophilic plasmid-mediated degradative capacity in a psychrotrophic bacterium.

A psychrotrophic bacterium, originally isolated from a natural aquatic environment, was characterized and identified as Pseudomonas putida Q5 for use as a representative recipient for biodegradative genes from a mesophilic microorganism. The TOL plasmid pWWO of the mesophile P. putida PaW1 was successfully transferred by conjugation to the naturally isolated psychrotroph P. putida Q5, as shown by plasmid analysis by agarose gel electrophoresis. Expression of the genes encoded by the mesophilic TOL plasmid in the psychrotroph was shown by the fact that the transconjugant (designated P. putida Q5T) had the capacity to degrade and utilize toluate (1,000 mg/liter) as a sole source of carbon at temperatures as low as 0 degrees C. Comparison of growth rates over a wide temperature range (0 to 30 degrees C) indicated that the physiological activity of the transconjugant was not reduced and that the plasmid DNA from the mesophile and its encoded enzymes functioned effectively in the psychrotroph at temperatures well below those at which the mesophile could grow. The production and demonstrated functioning of P. putida Q5T illustrates the possibility of developing specific degradative capacities in bacteria which can readily function at low temperatures in chemically contaminated environments or in industrial wastewater treatment systems.

Microbial decomposition of leaf material at 0°C.

The microbial decomposition of leaves (both fresh and autumnshed) at 0°C using stream sediment-water was investigated. The maximum rates of loss of leaf carbohydrate and protein at 0°C were considerable, being about 40% of those at 20°C. These rates were only slightly affected by the type of leaf material present being 1.3-fold higher with fresh leaves as compared with autumn-shed leaves. In addition, an epifluorescence microscopic counting technique was developed and utilized to enumerate the microbial populations colonizing the decomposing leaves. The average microbial densities on fresh and autumn-shed leaves after 35 days of incubation were 1.3 × 10(6) and 9.0 × 10(5) microorganisms cm(-2) at 0°C as compared with 5.5 × 10(6) and 3.3 × 10(6) microorganisms cm(-2) at 20°C, respectively. Antibacterial and antifungal antibiotics were used to estimate the comparative involvement of sediment bacteria and fungi in leaf degradation.

Effect of sulfide on nitrogen fixation in a stream sediment-water system.

Nitrogen fixation (C(2)H(2) reduction) in a sediment-water system was studied under anaerobic incubation conditions. Sodium sulfide at low concentrations stimulated activity, with a twofold increase in C(2)H(4) production occurring in the presence of 8 mumol of S per ml of stream water. Sodium sulfide at concentrations of 16 mumol of S per ml or greater inhibited nitrogen fixation, with 64 mumol of S per ml being completely inhibitory. Sulfide at levels of 16 mumol/ml or above inhibited CO(2) production, and the degree of inhibition increased with increasing concentration of sulfide. Titanium (III) citrate (used to modify Eh levels) stimulated both nitrogen fixation and CO(2) production, but could not duplicate, at any concentration tested, the twofold increase in nitrogen fixation caused by 8 mumol of S per ml. Sulfide additions caused pH changes in the sediment, and when the sediment was adjusted and maintained at pH 7.0 all concentrations of sulfide inhibited nitrogen fixation activity. From considerations of the redox equilibria of H(2), H(2)S, and other sulfur species at various pH values, it appeared that H(2)S was the toxic entity and that HS was less toxic. The observed stimulation of activity was apparently due to a pH change coupled with the concurrent production of HS from H(2)S.

Effect of sequence of exposure to chlorophenols in short-term bacterial bioassays.

A bioassay using Pseudomonas fluorescens was affected by the sequence of exposure to pentachlorophenol (PCP) and 2,3,4,5-tetrachlorophenol (TCP). Surviving cells from standardized cell suspensions initially treated with PCP at concentrations ranging from 10 to 75 micrograms/ml, followed by removal of the toxicant, were not affected by a second dose of PCP at the same concentration. However, if the second dose was TCP, the test organism was sensitive to the second exposure. The most toxic sequence was an initial exposure to TCP followed by a second exposure to PCP. The response of the test organism to PCP and TCP was clearly dependent upon both the toxicant concentrations used and the sequence of toxicant addition.

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.

Nitrogen fixation and methane metabolism in a stream sediment-water system amended with leaf material.

The reduction of acetylene and the production and oxidation of methane in a stream sediment--water system amended with either fresh leaves or autumn-shed leaves in the presence and absence of air were studied. Net methane production by the sediment--water system occurred only when leaf material was added, with fresh leaves giving 2.2 times the methane accumulation as autumn-shed leaves. Static incubation in the presence of air had little effect on net methane production, with such production being about the same as (with fresh leaves) or 79% of (with autumn-shed leaves) the anaerobic rates. Acetylene reduction was more affected by the presence of air, with anaerobic rates being at very low levels. The presence of fresh leaves was again stimulatory, with the acetylene reduction rate being 5.5-fold greater than that occurring with autumn-shed leaves.

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.

Effect of temperature on violacein production in a psychrotrophicChromobacterium from Lake Ontario sediment.

A truly psychrotrophic strain ofChromobacterium, which was isolated from Lake Ontario sediment and characterized asChromobacterium lividum, was found to be capable of pigment production that was completely prevented at 0°C, although growth readily occurred. Normal pigment formation occurred at 15°C, 20°C, and 25°C. The prevention of synthesis of the pigment at 0°C, which was confirmed spectrophotometrically to be violacein, was not reversed by the presence of various carbon sources, although all except one acted as growth substrates. In addition, some of the carbon sources actually inhibited pigment production at 20°C, preventing violacein synthesis in the presence of pyruvate which was shown to allow pigmentation. Similar results were obtained under both liquid and solid media cultivation conditions.

A method for the detection and analysis of growth patterns of microorganisms in soil.

A fluorescence-staining technique using the magnesium salt of 8-anilino-1-naphthalene sulfonic acid is described and used to follow the changes in the distribution patterns of microorganisms in soils. A statistical procedure was used to determine the occurrence of significant differences in clumping of bacteria (i.e., production of colonies) in different regions of artificial soil-aggregate systems treated with nutrient solutions and also with a herbicide, Linuron. The response of soil microorganisms to glucose amendment was most marked in the aerobic, outer zone of aggregates. Linuron inhibited colony formation in aggregates treated with the herbicide. The method allows continued observations to be made on the same soil sample at intervals during incubation and os can be used to determine growth rates, inhibitory effects of chemicals, distribution patterns in soils, effects of added nutrients, and other effects where growth in situ is important.

Effect of naturally occurring apatites on growth and morphology of algae.

Crystals (30--100 micrometer) of selected naturally occurring apatite (Ca10 (PO4)6(OH, F)2) samples were added to P-free (less than 0.001 microgram/ml total P) Bristol's medium (1-1000 microgram/ml of apatite) as the sole source of ortho-PO43-. The media were inoculated with washed, non-axenic cells of three chlorophycean algal species cultivated under PO43--deficient conditions. Phase-contrast and scanning electron microscopy revealed that at low slurry densities (1-10 microgram/ml of apatite), Ankistrodesmus braunii (ATCC 2744) cells were morphologically distorted. At concentrations of 100 and 1000 microgram/ml of apatite, more than 85% of the cells had undergone autospore formation within 7--10 days of incubation at 20 degrees C. Most autospores formed failed to germinate under high nutrient conditions. Scenedesmus longus (No. 1236) formed colonies when cultivated in Bristol's medium but daughter cells displayed a Chodatella-like unicellular morphology when grown in apatite media. Test algal species (Chlamydomonas dysosmos, S. longus, A. braunii) showed a marked preference for growth on apatite crystals over non-nutritive surfaces. Unialgal and mixed-algal cultures produced an extensive matrix of extracellular fibrous material in response to growth on crystals at concentrations greater than 10 microgram/ml of apatite.

A rapid, simple method for staining bacterial flagella.

A simple modification of Gray's flagellar staining procedure is described. It can be used on air-dried smears or directly on wet mounts of motile bacteria. The stained bacterial flagella can be observed with phase-contrast or bright-field optics. No rigorous cleaning of slides, counterstains, or any washing procedures are required with the staining method, making it very suitable for routine examinations.

Naturally occurring apatite as a source of orthophosphate for growth of bacteria and algae.

Several naturally occurring calcium-phosphate apatites which varied in crystalline structure and ionic composition were added as crystals of different particle size to P-free (<1µg/liter total P) nutrient media. Sufficient ortho-PO 4 (3-) was released by the partial dissolution of apatite crystals at limnetic pH levels (pH 7.8) to support growth of several unialgal-mixed bacterial cultures. The biomass produced by mixed populations increased as the amount of available apatite was increased and as the pH of the media and the particle size of the apatite crystals were decreased. These findings suggest that although apatite characteristically displays reduced solubility under alkaline conditions, the tons of apatite which are continuously entering aquatic environments as erosion material may be contributing to the P loading of those ecosystems.

Interactions between freshwater bacteria andAnkistrodesmus braunii in batch and continuous culture.

Batch and continuous cultures ofAnkistrodesmus braunii were established in an inorganic medium with growth rate limited by P. In batch culture, inoculation of lake water bacterial isolates ofPseudomonas sp. andFlavobacterium sp. showed that thePseudomonas isolate was capable of more rapid growth on algal exudates of lytic products than was theFlavobacterium isolate. When inoculated singly into a continuous culture (D=0.267 day(-1); P level, 2ΜM), theFlavobacterium isolate initially caused a decrease in the population density of the alga, but then steady states for both organisms were obtained. ThePseudomonas isolate under the same conditions caused a rapid washout of the algal culture, and steady-state conditions were never obtained. When thePseudomonas isolate was added to the two-member, steady-state system ofA. braunii andFlavobacterium, the algal population again washed out of the vessel, followed by theFlavobacterium and then thePseudomonas isolate. A transient increase in the P concentration to 200ΜM in the culture vessel caused the low algal population level to increase, followed by increases in the bacterial isolates when the algal population was high enough to supply the required organic carbon source. The system demonstrated that competition for P between the alga and the bacteria can occur, and the results were dependent on the algal and bacterial relative growth rates. The bacterial growth rates were limited initially by organic substrates produced by the alga, and the different bacterial isolates competed for these substrates.