Protectants used in the cryopreservation of microorganisms

Publisher Summary This chapter focuses on the culturing methods for cyanobacteria. For the establishment of new cultures, as much ecological information as possible about the field populations should be gathered and recorded. With this, it eventually becomes possible to relate diverse information obtained from culture strains to the survival strategies of the organism in nature, ultimately the object of all biological research unless it is directed solely to the benefit of humans. Cyanobacteria, unlike most other prokaryotes (eubacteria or archaebacteria) are usually seen and often recognizable (even at the species level) before collection; they are present in many cases as plankton blooms or as dense tufts or mats which are composed of few other species. Only a miniscule percentage of cyanobacterial species have been brought into culture. This, in part, reflects the relatively few and mainly recent attempts to isolate and grow diverse cyanobacteria. Cyanobacteria from certain habitats and those of certain taxonomic groups seem to be extremely recalcitrant with regard to being cultured with present methods. Identification of cyanobacteria, whether of natural populations or of cultured material, is not a simple task, because at present, there are three major systems of classification: Geitler, Drouet, and Stanier systems.

[3] Culturing methods for cyanobacteria

A thermodynamic framework has been provided for the description of maintenance requirements of microorganisms. The central parameter is the biomass specific Gibbs energy consumption for maintenance, m(E) (kJ/C-mol biomass . h). A large set of data has been used including (i) a large range of different organisms (bacteria, yeasts, plant cells), (ii) mixed cultures, (iii) heterotrophic and autotrophic growth, (iv) growth under aerobic and anaerobic conditions, and (v) a large temperature range (5-75 degrees C). It appears that only the temperature has a major influence, with an energy of activation of 69 kJ/mol. Different electron donors or electron acceptors only show a very minor influence on m(E). On the basis of the data set, temperature correlations of m(E) have been derived for aerobic and anaerobic growth. The generalized concept for maintenance Gibbs energy is used to establish a correlation which allows the estimation of the biomass yield on electron donor as a function of C-source, electron donor, electron acceptor, N source, growth rate, and temperature. The advantage of using the m(E) parameter over other maintenance-related parameters (like mu(e), m(O2), m(D), gamma(D)m(D)) is discussed.

A thermodynamically based correlation for maintenance gibbs energy requirements in aerobic and anaerobic chemotrophic growth.

Carboxymethyl-cellulase and β-glucosidase activities were determined in the cytosole, cell walls and extracellular culture fluid of Trichoderma reesei QM 9414 cultivated on cellulose and cellobiose. By means of carboxymethylcellulose as a specific desorbens for cellulose bound CM-cellulase and β-glucosidase it was found that these enzymes are cell wall bound during consumption of the carbon source, but are excreted during the subsequent cultivation stage. Treatment of intact cell walls with various chemical agents could not cause a release of the enzyme. Treatment of intact cell walls with α-mannanase or trypsin released CM-cellulase, whereas, treatment with laminarinase or chitinase released β-glucosidase. Both enzymes were also released during autolysis in phosphate buffer. This autolysis was accompanined by the appearance of extracellular mannanase, laminarinase and proteinase. The results suggest that cleavage of chemical bonds of certain cell wall polymers of T. reesei could be responsible for the appearance of CM-cellulase and β-glucosidase in the culture fluid during later stages of growth.

Release of carboxymethyl-cellulase and β-glucosidase from cell walls of Trichoderma reesei

Publisher Summary This chapter discusses various methods used to preserve microorganisms. Storage in liquid nitrogen is one of the best methods for preserving all microorganisms. For some viruses, it may be better to freeze the sample rapidly, but slow freezing with a cryoprotective agent is desirable for all other organisms to retain maximum viability or infectivity. Storage at –70°C is nearly as effective as –196°C (liquid nitrogen). Only a few organisms survive well at higher temperatures, such as –20 or –40 ° C. For short-term preservation (3–12 months), agar slants or stab cultures in tightly capped tubes are adequate. For long-term storage of those organisms that can withstand dehydration, lyophilization is the most economical and reliable method. Although many lyophilized organisms survive well at room temperature, all cultures should be refrigerated. Certain problems associated with lyophilized cultures must be recognized. Dehydration may be mutagenic and, in addition, the relative number of spontaneous mutants can increase in the rehydrated culture.

Preservation of Microorganisms

The enzyme-forming systems (EFSs) concerned with the amylase of Bacillus subtilis, and the glucamylase and acid protease of Aspergillus niger, have been shown to be highly stable in the cultural conditions applied owing to the low decay rate of mRNAs specific for these respective enzymes. The quantity per cell of mRNAs for any of these enzymes is regarded as limiting the specific rate of enzyme production in the conditions used for enzyme production. Investigations on the repression, derepression and the preferential synthesis of these hydrolases have led to simple hypothetical relationships.

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Kinetics of hydrolase production by microorganisms

Freezing and freeze-drying of Spirulina platensis

Bacterial cells are separated from a culture broth by adding thereto a solution of casein or alkali-soluble soybean proteins, said culture broth being maintained at a pH less than 6. Bacteria cells are flocculated to form a protein-bacteria co-precipitate which is easily separable by a conventional procedure. The co-precipitate may be directly put to uses such as for foods or feeds.

Method for separation of bacteria cells from culture broth

Kinetic Studies on Enzyme Production by Mictobes : (III) Process Kinetics of Glucamylase Production by Aspergillus niger

Preferential Synthesis of Extracellular Glucoamylase by Aspergillus niger : Stability of mRNA Associated with Membrane-boud Polysomes in Nongrowing Mycelia

Gyozo Terui

Papers

Kinetics of hydrolase production by microorganisms

Freezing and freeze-drying of Spirulina platensis

Method for separation of bacteria cells from culture broth

Kinetic Studies on Enzyme Production by Mictobes : (III) Process Kinetics of Glucamylase Production by Aspergillus niger

Preferential Synthesis of Extracellular Glucoamylase by Aspergillus niger : Stability of mRNA Associated with Membrane-boud Polysomes in Nongrowing Mycelia