Growth of Mycobacterium rhodochrous on n‐Decane: a New Growth Factor and Emulsifying Agent

Abstract

During the study of the growth of Mycobacteriurn rhadochnous NCIB 9905 in a minimal medium with n-decane as the sole carbon source, a growth factor and a hydrocarbon emulsifying factor were discovered. The factors weie heat stable, were released from cells by autolysis or by physical disruption and functioned independently of one another. Inoculations of < 10' cells/ml of culture medium routinely failed to grow unless an autoclaved cell suspension or purified extract was added. This growth factor requirement was not satisfied by exogenous trace metals, vitamins, amino acids, intermediates of decane metabolism or Mycobactin, but was satisfied by an extract from cells of an unrelated species growing on n-decane. The extract of strain 9905 did not satisfy the sideramine requirement of Arthrobactev JG9 and was not associated with ferric ions. Some evidence was obtained suggesting that the growth factor may be of a new type involving the chelation of calcium ions. DURING THE ISOLATION of hydrocarbon-oxidizing organisms from various soil and water samples it was clear that the predominant bacteria belonged to morphologically related groups including Mycobacterium, Arthrobacter and Nocardia. Most of the strains exhibited considerable variation in daily subcultures and often lost the rapidity of growth observed when originally isolated. These variations included passage of a large proportion of the culture from the smooth tb the rough phase, as shown by the dry wrinkled appearance of isolated colonies on agar media and by the appearance of pellets of cells in liquid culture. The pellets reached 5-10 mm in size and posed a serious problem in the industrial exploitation of the bacteria concerned. With one strain, Mycobacterium rhodochrous NCIB 9905, the inability to initiate growth in a mineral salts medium unless large inocula were used was particularly striking, and the nature of the phenomenon was investigated. Materials and MethodsCultures The hydrocarbon-utilizing organisms used were isolated from spillage area soils in the Oil Works of I.C.I. Heavy Organic Chemicals Division, Billingham, Co. Durham, using enrichment cultivation methods and the medium and carbon source described below. Two specimen cultures were deposited in the National Collection of Industrial Bacteria and designated respectively as Pseudomoms aeruginosa (NCIB 9904) and Myco. rhodochrous (NCIB 9905).

Sequential Growth of Bacteria on Crude Oil

“…Microorganisms can utilize crude oil as a substrate for growth with or without concomitant oil emulsification (1,15). Although there have been some attempts to characterize the emulsifying agents, the preparations have not been purified sufficiently to identify the active components (12,18). In the present study oil emulsification was obtained during the exponential growth phase with UP-2, stationary phase with RAG-1, and not at all with UP-3 and UP-4.…”

Emulsifier of Arthrobacter RAG-1: isolation and emulsifying properties

“…Below 1.5 mg of gas oil per ml, turbidities were directly proportional to gas oil concentration. At between 8 and 30 mg of gas oil per ml, turbidity increased about 5 Klett units/mg of gas oil.…”

“…V 400 I GAS-OIL (mg/mI) FIG. 5. EF-RAG-induced emulsion formation as a function ofgas oil concentration.…”

Effectiveness of surfactants in the microbial degradation of oil

“…Detergents are surface-active agents (surfactants) that increase the oil-water interfacial area by emulsification, and, thus, might be expected to stimulate the microbial degradation of the hydrocarbons. Indeed, many microorganisms are known to produce surfactants when grown on hydrocarbons as a sole source of carbon and energy (Gerson et al, 1976;Zajic and Supplison, 1972;Holdom and Turner, 1969). Different surfactants have various effects on the utilization and breakdown of hydrocarbons by microorganisms (Bertrand et al, 1976;Whitworth et al, 1973;Robichaux and Myrick, 1972).…”