Several conditions of growth of Bordetella pertussis cause a reversible phenotypic alteration in properties termed modulation. Growth in medium containing nicotinic acid induces normal (X-mode) cells to change to modulated (C-mode) cells. We examined several pyridines and compounds resembling pyridines for their ability to affect modulation, using envelope protein patterns and serological reactivity as indicators of modulation. We found that 6-chloronicotinic acid and quinaldic acid were more effective modulating stimuli than was nicotinic acid on a molar basis. Both 2-chloronicotinamide and isoniazid interfered with nicotinic acid-induced modulation, and can be called antimodulators. Picolinic acid inhibited growth.

Mucinase from Vibrio cholerae strain CA401 was purified by precipitation with ammonium sulfate and chromatography on a column of BioGel P-100 (Bio-Rad Laboratories, Richmond, California). Ovomucinase, intestinal mucinase, and protease appeared as two peaks of slightly different molecular weights, comigrated in nondenaturing polyacrylamide gel electrophoresis, demonstrated similar patterns of inhibition by heavy metals, and were inhibited by antiserum to purified mucinase. Both molecular weight forms exhibited similar properties, which were identical to those of fresh culture supernatants. Specific activity was only slightly increased by purification. Antiserum to mucinase passively protected infant mice from diarrhea due to V. cholerae.

Mutants of Vibrio cholerae that were deficient in protease production were isolated by picking clones form gelatin or casein plates which showed reduced zones of proteolysis. All mutants showed reduced ability to degrade complex proteins (casein and gelatin), and those tested were deficient in ability to degrade chicken egg ovomucin. Some of the mutants demonstrated a decrease in neuraminidase activity. Almost all mutants showed a dramatic loss of virulence in the infant mouse, although toxin was still produced. A partial revertant, to protease-proficient, demonstrated a simultaneous increase in neuraminidase activity and also an increase in mouse virulence. The strains described had a variety of phenotypes.

The field history and performance of microbial culture products for the oil field is examined. For over 15 years, microbial culture products have been used for paraffin control, production enhancement, well bore treatments as well as for scale and corrosion problems. The wide-ranging capacity of microbes to effect positive changes in oil and water properties is described. The broad spectrum of oil types and formations that have been treated successfully is reported along with treatment protocols. Mechanistic considerations for modes of action are analyzed. Traditionally, these considerations involve the continuous production of biosurfactants, solvents and other oil mobilizing agents. Continuous advancement of microbial technology has led to more recent development of new applications that use unique metabolic capabilities of microorganisms to address specific well problems. Examples of applying these products to problems in oil field production systems are shown. The outlook for development of new technologies and the future application of these products to the oil field is discussed. Introduction Microbial culture products occupy an increasingly important and growing segments in oil field production operations. They are a truly environmentally benign treatment technology that can be used to replace and augment many conventional technologies, including many oil field chemicals. The extraordinary diversity of microorganisms with the concomitant likelihood for many more such products in the future suggests that their role in oil field operations will continue to expand and will supplant many conventional technologies in the next 100 years. It is therefore important to review the prior and current uses of this technology. Historical Applications of Microbial Culture Products Paraffin Control. Microbial culture products (MCPs) were first used in 1986 in the Austin Chalk formation in Texas to control paraffin deposition. The theory behind these products was that microorganisms can be isolated and combined in novel mixtures which will produce biochemicals that will mimic the action of classic oil field chemicals such as pour point depressants, crystal modifiers and wax dispersants. The advantage of using such biological products is the fact that the microorganisms will 1) produce these biochemicals continuously and 2) attach to surfaces where paraffin deposition is occurring and act directly at the site of deposition. The first successful application of these products began a pattern of expansion that continued throughout the 80s and 90s. Paraffin deposition results in a variety of problems for oil field operators, ranging from plugging of tubulars to occult formation deposition that reduces formation permeability. A continual increase in the number of products available to the industry allowed the expansion of the microbial technology for paraffin control into a variety of different oil types and formations. Conventional technologies to control paraffin deposition are thermal and chemical treatments. Both of these technologies have limitations that restrict their long-term effectiveness. In particular, hot oil or water treatments may lead to increased formation damage by forcing deposited high molecular weight paraffins into the formation where they can contribute to pore throat plugging and lead to production loss. Development of MCPs represents a successful alternative technology to remove paraffin deposits without causing lasting formation damage. Long term use of MCPs showed no damage to the oil field production system and their use increased throughout the mid continent region in the early 1990s. Examples of the successful application of this technology in the oil field have been previously documented in SPE papers.1,2,3

A simple method for the analysis of microbial proteases is described that was used to characterize the proteolytic activities of various Vibrio cholerae isolates. This method utilized the unique peptides generated from the degradation of a standard protein by proteases of various specificities. These peptides were anaâ lyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The unique patterns of peptides seen in gels can be used to type proteases according to their relative specificities. Culture supernatants of V. cholerae isolates from a variety of environmental and human sources were analyzed for the presence of a protease previously isolated and characterized in this laboratory from V. cholerae strain CA401. Supernatants from most isolates showing dimethyl casein proteolytic activity exhibited the presence of enzymes similar to the CA401 protease in their peptide digest patterns against bovine serum albumin and in their immunological reactivities. The probable widespread presence of this virulence-associated protease in V. cholerae isolates is discussed.