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The soluble and dispersed nitrogen and carbon components in the filtrate fraction of cattle feedlot waste are a potential nutrient source from which single-cell protein could be produced for animal feeds. The ability of more than 200 fungi and streptomycetes to grow in this liquid was determined; these included isolates from the waste and associated sources, as well as organisms maintained in the Culture Collection of the Agricultural Research Service in Peoria, Ill. Utilization of waste nutrients was measured by changes in nitrogen content and chemical oxygen demand. Only 20% of the organisms were able to grow appreciably in the filtrate. Of these, dry-weight yields varied from 0.6 to 2.7 g of mycelium per liter; from 21 to 50% of the nitrogen in the filtrates was used during growth, whereas chemical oxygen demand levels diminished from 4 to 60%. In general, streptomycetes isolated from the feedlot used nutrients from the filtrates better than fungi did. Addition of readily available carbon sources such as glucose or whey significantly increased (as much as sixfold) cell yields of selected organisms and promoted better utilization of nitrogen (from two- to threefold); the effect on chemical oxygen demand varied (0 to 33% increase).

Filtrates from feedlot waste were incubated under aerobic conditions to evaluate the availability of nutrients for cell production and to assess the capacity of indigenous flora to produce stabilized effluents. Incubation was carried out in 9-liter aerated jar fermentors. Three-fourths of the organic material and one-third of the nitrogen were taken up in 4 days; 90% utilization of organic material and nitrogen required almost a month. Acid was produced initially, but aerated liquid thereafter rapidly became alkaline. With pH controlled at 7.0, a comparable pattern of carbon utilization occurred, but nitrogen was incompletely used. The numerically dominant organisms in the waste inoculum were almost immediately displaced by an emergent population of a few types of organisms not originally evident. Maximal viable populations of 109to 3 � 109cells/ml were obtained in aerated waste liquid within 48 h; subsequently, numbers declined quickly to initial levels. Numbers of fungi, yeasts, and streptomycetes slowly increased but never exceeded their initial concentration by more than tenfold.

Growth characteristics of the insect pathogen, Bacillus popilliaeDutky, were studied by propagation in shaken flasks and in 2-liter fermentors. Maximal populations between 5 × 108and 2 × 109viable cells per milliliter of culture medium routinely were obtained in incubation periods of 18 to 24 hr at 30 C in a medium composed of 1.5% yeast extract, 0.6% K2HPO4, and 0.2% glucose or trehalose. The carbohydrate required for growth in liquid media was fermented with the formation of 2 meq of acid per mmole of carbohydrate utilized; acid products ordinarily were not subsequently metabolized. B. popilliaeis an aerobe, and the amount of growth obtained varied with aeration to an optimum at oxygen absorption rates of about 0.5. Maximal populations persist in a culture for periods of only 1 to 4 hr; cessation of growth was followed immediately by rapid death of cultures, so that less than 1% of the cells remained viable after 48 hr, and viability often was lost entirely by the end of 72 hr of incubation. No cytological evidence for spore formation was observed under any growth condition. Death was not associated with lysis of the cells, although extensive granulation ultimately occurred. Continuous neutralizaiton, augmented buffering, various techniques of dialysis, or slow feeding of the carbohydrate did not markedly alleviate the characteristic death of the cultures.

Physical properties and chemical structure of two related polysaccharides produced fermentatively by Plectania occidentalisNRRL 3137 and by Helotiumsp. NRRL 3129 were studied. Both polymers were readily recovered as amorphous gels by precipitation from culture liquors with two parts of ethyl alcohol or methanol. Dried polymeric material was redissolved in water with agitation to give uniform aqueous solutions up to about 1.5% by weight. The polymers were similar in physical properties but possessed different chemical structures. The viscosity of aqueous solutions of each polymer varied from about 50 centipoises at 0.1% to approximately 2,200 centipoises at 1.4% concentrations by weight. Highly viscous solutions at concentrations of 1% or greater behaved like thixotropic gels. Mono-, di-, and trivalent salts, except borate, did not affect viscosity of either polymer. The viscosities were slightly increased by the addition of borate. Autoclaving did not alter the physical properties of neutral polymer solutions. The polymers were stable in acid or alkaline solutions at moderate temperatures but degraded under extremes of pH at 70 C or above. Each polymer had a specific rotation of +20° in aqueous dimethylformamide (1:1). The results of acid hydrolysis and periodate oxidation, in conjunction with paper and gas chromatography, indicate that both polymers are branched glucans containing appreciable amounts of β-1,3 linkages.

Two specimens of higher fungi produced exocellular β-1, 3-glucans when their mycelial forms were cultivated under submerged aerobic conditions. Plectania occidentalisNRRL 3137 consumed up to 6% glucose or xylose with about 30% conversion to polymer in a medium composed of hydrolyzed soy protein, salts, and thiamine. A 5% inoculum was used in a 10-day shaken fermentation. After dilution of the culture liquors and partial disruption of mycelia with a blender, solids were removed by centrifugation, and the polymer was precipitated by the admixture of 2 volumes of ethyl alcohol. A second polymer was formed in 40 to 65% yield by fermentation with Helotiumsp. NRRL 3129, which in the imperfect stage would be identified as Moniliasp. It consumed up to 4% glucose, fructose, mannose, or sucrose in 60 to 72 hr. A 2% inoculum in a medium composed of commercial defatted soy flakes, phosphate, and thiamine in tap water gave a satisfactory fermentation. This polymer was precipitated by the addition of 0.5 volume of ethyl alcohol. Both organisms have a broad pH optimum on the slightly acidic side and did best at about 25 C.

The conditions necessary for the production of fumaric acid in 20-liter fermentors by fermentation of glucose with Rhizopus arrhizusstrain NRRL 2582 were determined. Continuous neutralization of fumaric acid was necessary for optimal yields. Yields of the calcium salt were in excess of 65 g of fumaric acid from 100 g of sugar consumed during fermentation of sugar concentrations of 10 to 16%. Conditions established for calcium fumarate production include a simple mineral salts medium, 0.5 v:v:min aeration rate, 300 rev/min agitation rate in a baffled tank, 33 C incubation temperature, CaCO3to neutralize the acid formed, and a 4 to 5% (v/v) vegetative inoculum. A suitable procedure and medium for the preparation of a vigorous vegetative inoculum were established. The tendency for calcium fumarate fermentations to foam excessively was controlled with a proper antifoam agent added prior to sterilization of the medium and again at daily intervals during fermentation. The production of soluble sodium or potassium fumarates was inhibited when the concentration of fumarates reached 3.5 to 4.0%. No means of overcoming this inhibition was found. Starches and certain other grain-derived carbohydrates were fermented to form calcium fumarate in flask experiments with approximately the same efficiency as was glucose.

Samples of beef cattle feedlot waste (FLW), runoff from the pens, and water from a large drainage ditch at the feedlot were examined for Enterobacteriaceae. The drainage ditch receives the runoff but contains primarily subsurface drainage from fields on which FLW is spread for disposal. Planting and enrichment techniques with seven different media were used to isolate 553 cultures of enterobacteria. FLW contains about 50 million enterobacteria/g dry weight. More than 90% of these were Escherichia coli, none of which were enteropathogenic types as determined with multivalent sera. Citrobacterand Enterobacter cloacaewere other organisms present in moderate numbers. Application of enrichment techniques broadened the spectrum of enterobacteria isolates to include the four Proteusspp., both Providenciaspp., Klebsiella, Enterobacter aerogenes, Arizona, and a single isolate of Salmonella(serological group C2). Shigellawas not isolated. The wide spectrum of enterobacteria in FLW may be a hazard if unsterilized waste is refed. Fewer enterobacteria occurred in the runoff and in the drainage ditch; the most numerous species in FLW also were most numerous at these sites. However, neither Salmonellanor Arizonawas isolated from runoff or drainage-ditch waters.

A quantitative determination was made every 2 months for a year of the microflora of beef cattle waste and runoff at a medium-sized midwestern feedlot. Counts were obtained for selected groups of organisms in waste taken from paved areas of pens cleaned daily and, therefore, reflect the flora of raw waste. Overall, in terms of viable count per gram dry weight, the feedlot waste contained 1010total organisms, 109anaerobes, 108gram-negative bacteria, 107coliforms, 106sporeformers, and 105yeasts, fungi, and streptomycetes. The specific numbers and pattern of these groups of organisms varied only slightly during the study in spite of a wide variation in weather. Data indicate that little microbial growth occurs in the waste as it exists in the feedlot. Runoff from the pens contained the same general population pattern but with greater variation attributable to volume of liquid. Comparable determinations of an associated field disposal area (before and after cropping), stockpiled waste, and elevated dirt areas in the pens indicate that fungi, and especially streptomycetes, are the aerobic organisms most associated with final stabilization of the waste. Yeasts, which are the dominant type of organism in the ensiled corn fed the cattle, do not occur in large numbers in the animal waste. Large ditches receiving runoff and subsurface water from the fields have a population similar to the runoff but with fewer coliforms.

The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast microscopy. Although fine surface detail is not elucidated by scanning electron microscopy, this technique does afford a definitive picture of the general shape of spores. Spores of Bacillus popilliae, B. lentimorbus, B. thuringiensis, B. alvei, B. cereus,and Sarcina ureaehave varying patterns of surface ridge formation, whereas spores of B. larvae, B. subtilis,and B. licheniformishave relatively smooth surfaces.