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The lipopolysaccharides (LPS) of the obligate acidophile Thiobacillus ferrooxidans grown on iron, sulfur, and glucose as energy sources were examined for various physical and chemical properties. Both qualitative and quantitative variations were found among the three preparations. The LPS extracted from iron-grown cells (Fe-LPS) contained less than 3% protein compared to 18 to 25%c in LPS extracted from either sulfur-grown cells (S-LPS) or glucose-grown cells (G-LPS). S-LPS showed two distinct sedimentable species. 61S and 9.3S, which could be fractionated on a column of Sepharose 4B. The relative densities of' both S-LPS and G-LPS were found to be significantly greater than that of Fe-LPS. Spectral differences were noted when each LPS was reacted with a carbocyanine dye. Fe-LPS showed a single absorbance maximum at 472 nm, S-LPS displayed its maximum at 650 nm, and G-LPS showed two maxima, the first at 468 nm and the other at 655 nm. Analysis of the methyl ester derivatives of the LPS t'atty acids using gas chromatography-mass spectrometry revealed the presence of' a very stable species, tentatively identified as a methoxy methyl ester with a formula of CH3-O-C10H10-COOCH3, as the major component from each LPS. 3-Hydroxymyristic acid was found only in Fe-LPS.

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A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 330C; pH, 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specified by the Food and Agricultural Organization of the United Nations.

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehydeor methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of Cl compounds. Its Cloxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle. This medium was designed to select for hyphomicrobia and other methylotrophs. Cultures were originally inoculated with raw sewage 56