I Mill I1MENT LIQUID CULTURE
liiiiclinient liquid culture is frequently carried out in shake flasks. However, the growth of the desired type from a mixed inoculum will result in the modifica-timi ol llie medium and therefore changes the selective force which may allow the growth of other organisms, still viable from the initial inoculum, resulting in a succession. The selective force may be re-established by inoculating the enriched culture into identical fresh medium Such sub-culturing may be repeated several times before the dominant organism is isolated by spreading a small inoculum of the enriched culture onto solid medium. The time of sub-culture in an enrichment process is critical and should correspond to lho point .it which the desired organism is dominant.
The prevalence of an organism in a batch enrichment culture will depend on its maximum specific growth rate compared with the maximum specific growth rates of the other organisms capable of growth J" the inoculum. Thus, provided that the enrichment uioth is sub-cultured at the correct times, the dominant organism will be the fastest growing of those capable of growth. However, it is not necessarily true that the organism with the highest specific growth rate is the most useful, for it may be desirable to isolate the organism with the highest affinity for the limiting substrate.
The problems of time of transfer and selection on the basis of maximum specific growth rate may be overcome by the use of a continuous process where fresh medium is added to the culture at a constant rate. Under such conditions the selective force is maintained at a constant level and the dominant organism will be selected on the basis of its affinity for the limiting substrate rather than its maximum growth rate.
The basic principles of continuous culture are considered in Chapter 2 from which it may be seen that the growth rate in continuous culture is controlled by the dilution rate and is related to the limiting substrate concentration by the equation:
Equation (3.1) is represented graphically in Fig. 3.1. A model of the competition between two organisms capable of growth in a continuous enrichment culture is represented in Fig. 3.2. Consider the behaviour of the two organisms, A and B, in Fig. 3.2. In continuous culture the specific growth rate is determined by the substrate concentration and is equal to the dilution rate, so that at dilution rates below point Y in Fig. 3.2 strain B would be able to maintain a higher growth rate than strain A, whereas at dilution rates above Y strain A would be able to maintain a higher growth rate. Thus, if A and B were present in a continuous enrichment culture, limited by the substrate depicted in Fig. 3.2, strain A would be selected at dilution rates above Y and strain B would be selected at dilution rates below Y. Thus, the organisms which are isolated by continuous enrichment culture will depend on the dilution rate employed which may result in the isolation of
Residual substrate concentration
Fig. 3.1. The effect of substrate concentration on the specific growth rate of a micro-organism.
Residual substrate concentration
Fig. 3.1. The effect of substrate concentration on the specific growth rate of a micro-organism.
organisms not so readily recovered by batch techniques.
Continuous enrichment techniques are especially valuable in isolating organisms to be used in a continuous-flow commercial process. Organisms isolated by batch enrichment and purification on solid media frequently perform poorly in continuous culture (Harrison et al., 1976), whereas continuous enrichment provides an organism, or mixture of organisms, adapted to continuous culture. The enrichment procedure should be designed such that the predicted isolate meets as many of the criteria of the proposed process as possible and both Johnson (1972) and Harrison (1978) have discussed such procedures for the isolation of organisms to be used for biomass production. Johnson emphasized the importance of using the carbon source to be employed in the subsequent commercial process as the sole source of organic carbon in the enrichment medium, and that the medium should be carbon limited. The inclusion of other organic carbon sources, such as vitamins or yeast extract, may result in the isolation of strains adapted to using these, rather than the principal carbon source, as energy sources. The isolation of an organism capable of growth on a simple medium should also form the basis of a cheaper commercial process and should be more resistant to contamination — a major consideration in the design of a commercial continuous process. The use of as high as possible an isolation temperature should also result in the isolation of a strain presenting minimal cooling problems in the subsequent process.
The main difficulty in using a continuous-enrichment process is the washout of the inoculum before an adapted culture is established. Johnson (1972) suggested that the isolation process should be started in batch culture using a 20% inoculum and as soon as growth is observed, the culture should be transferred to fresh medium and the subsequent purification and stabilization of the enrichment performed in continu. ous culture. The continuous system should be pcriodj. cally inoculated with soil or sewage which may not only be a source of potential isolates but should also ensure that the dominant flora is extremely resistant to con tamination.
Harrison (1978) proposed two solutions to the problem of early washout in continuous isolation processes5 The first uses a turbidostat and the second uses a two-stage chemostat (see Chapter 2). A turbidostat is a continuous-flow system provided with a photoelectric cell to determine the turbidity of the culture and maintain the turbidity between set points by initiating or terminating the addition of medium. Thus, washout is avoided as the medium supply will be switched off f the biomass falls below the lower fixed point. The use of a turbidostat will result in selection on the basis of maximum specific growth rate as it operates at high: levels of limiting substrate. Thus, although the use of the turbidostat removes the danger of washout it is not as flexible a system as the chemostat which may be used at a range of dilution rates. The two-stage chemostat described by Harrison (1978) is very similar to Johnson's (1972) procedure. The first stage of the -.<» tem was used as a continuous inoculum for the second stage and consisted of a large bottle containing a basic medium inoculated with a soil infusion. Continuous inoculation was employed until an increasing absor-bance was observed in the second stage. Bull (1992) advocated the use of feed-back continuous systems for the isolation of strains with particularly high affinity for substrate and this approach would also guard against premature washout.
The use of continuous enrichment culture has frequently resulted in the selection of stable, mixed cultures presumably based on some form of symbiotic relationship. It is extremely unlikely that such mixed," stable systems could be isolated by batch techniques so that the adoption of continuous enrichment may result in the development of novel, mixed culture fermentations. Harrison et al. (1972) isolated a mixed culture using methane as the carbon source in a continuous-enrichment and demonstrated that the mixture contained one methylotroph and a number of non-methyl-otrophic symbionts. The performance of the methylotroph in pure culture was invariably poorer than the, mixture in terms of growth rate, yield and culture^ stability. :
Continuous enrichment has also been used for the isolation of organisms to be used in systems other than miilluiMliHK K™^ a',d BU" (197?) r' ^ bionwf" P",u . iu. an Anhrobacter sp. producing a technic 10 |Sl ' , „ mp|cx. The technique has been vcast Ivsinj: ci1/>|M- - i
UTl i.t'i -d media have been used for the isolation of n/vmc pioJucers and these techniques usually CCIIi!,n fh -'use ol .1 -elective medium incorporating the V ni ihe .-n/vmc which encourages the growth AunstruP * * am *****d ol l<«<producing alkaline proteases. Soils TMmous nil- "cic used as the initial inoculum and, V ■ -it tin cMent. the number of producers isolated
T,r"'-uu-d ''lk»lini,yof thc soil sample-The soil' samples wee ,,.steurized to eliminate non-sporu-,,till„ o.'-a...s,ns and then spread onto the surface of ■,'mi'"media at pi I "-HI. containing a dispersion of an insoluble ptulein Colonics which produced a clear zone due to thc digestion of the insoluble protein were taken ic lv alkaluu protease producers. The size of the dcaiinn /one uuild not be used quantitatively to select Inch piodikvis as there was not an absolute correlation between the m/c .'I the clearing zone and the production ol alkaline pi mease in submerged culture. How-cvei. this example demonstrates the importance of choice ot siaitin;.' material, thc use of a selective force in the isolation .md the incorporation of a preliminary diagnostic tcsi. albeit of limited use.
Isiilaliun methods not utilizing selection of the desired characteristic
'lhe s\ nthcsis oi some products does not give the pioducing niiianisin any selective advantage which may be exploited directly in an isolation procedure. Examples include the production of antibiotics and growth prumoteis. Ihcicloie, a pool of organisms has to be isolated and subsequently tested for the desired charac-tenstic |"he nia|ni problem faced by industrial microbiologists in this situation is the reisolation of strains which have ,ihcad\ been screened many times before. 1 lowevei. this 'ivimention of the wheel' syndrome may be mininu/cd in tuc major ways:
ID l)e\clopmg procedures to favour the isolation of unusual taxa which are less likely to have been screened previously.
(ii) Identifying selectable features correlated with the unselectable industrial trait thus enabling an enrichment process to be developed.
Much use has been made of numerical taxonomic data bases to design media selective for particular taxa. For example, Williams' group at Liverpool University (U.K.) used such databases to design isolation media to either encourage the growth of uncommon strep-tomycetes or to discourage the growth of Streptomyces albidoflavus (Vickers et al, 1984; Williams and Vickers, 1988). Several groups of workers have taken advantage of the antibiotic sensitivity information stored in taxonomic databases to design media selective for particular taxa, as shown in Table 3.2. The incorporation of particular antibiotics into isolation media may result in the selection of the resistant taxa. Such techniques are reviewed by Goodfellow and O'Donnell (1989) and Bull (1992). Bull emphasized that the taxonomic approach may be optimized and developed according to Fig. 3.1. The isolates from an isolation procedure would be screened for activity and then the growth requirements of the positive cultures determined. This knowledge can then be used to optimize the isolation medium and the cycle begins again.
Whilst taxonomic databases are a convenient source of information, it is important to appreciate Huck et al's (1991) observation that these systems were designed to provide information for taxonomic differentiation within a group. Thus, some of the diagnostic data may not be applicable to isolation systems. More significantly, the reactions of organisms outside the taxon in question would not be listed. Of course, an environmental sample contains a vast variety of organisms and the design of isolation media based on knowledge of only one taxon may inadvertently result in the preferential isolation of undesirable types. Huck et al. (1991) used the statistical stepwise discrimination analysis (SDA) technique to design media for the positive selection of antibiotic producing soil isolates. This was achieved by characterizing a collection of eubacte-rial and actinomycete soil isolates according to 43 physiological and nutritional tests. Certain features were identified which, when used as selective factors, enhanced the probability of either isolating acti-nomycetes or antibiotic producing actinomycetes. These features are shown in Table 3.3. Using this approach several media were developed which enhanced the isolation of antibiotic producers.
The most desirable isolation medium would be one
Table 3.2 Antibacterial compounds used in selective media for the isolation of actinomycetes (Goodfellow and O'Donnell, 1989)
Preobrazlienskayai et al. (1975)
Tomita et al. (1980) Bibikova et al. (1981) Chormonova (1978) Yoshioka (1952) Sveshnikova et al. (1976) Willoughby (1971) Wakkisaka et al. (1982)
which selects for the desired types and also allows maximum genetic expression. Cultures grown on such media could then be used directly in a screen. However, it is more common that, once isolated, the organisms are grown on a range of media designed to enhance productivity. Nisbet (1982) put forward some guidelines for the design of such media and these are summarized in Table 3.4.
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