Inoculum stages

Optimisation of biomass production would require a large inoculum, comprising 10% of each inoculum stage. However, this involves many transfers which increases the risk of contamination. 

The inoculum stage media are designed to provide the organism with all the nutrients that it requires. Adequate oxygen is provided in the form of sterile air and the temperature is controlled at the desired level. Principal criteria for transfer to the next stage in the progression are fieedom from contamination and growth to a pre-determined cell density. 

Step 4 Manufacturing scale-up Inoculum stage Fermentation or cell culture stage Protein purification stage Formulation stage... 

A commercial technology (69), the SABRE process, treats contaminated water and soil ia a two-stage process by adding a readily degradable carbon and an inoculum of anaerobic bacteria able to degrade the contaminant. An initial aerobic fermentation removes oxygen so that the subsequent reduction of the contaminant is not accompanied by oxidative polymerization. 

Inoculum Preparation Stage Two batches of inoculum of about 50 gallons each are prepared by the following method A 25 ml inoculum (from the germination stage) is transferred to each of four 2-liter flasks, each containing 500 ml of the sterile medium utilized for germination. The flasks and contents are incubated for 5 days at 28°C on a rotary shaker (280 rpm, 2 inch stroke). 

For economical reasons the fermentation time should be as short as possible with a high yield of the amino acid at the end. A second reason not to continue the fermentation in the late stationary phase is the appearance of contaminant-products, which are often difficult to get rid off during the recovery stage. In general, a relatively short lag phase helps to achieve this. The lag phase can be shortened by using a higher concentration of seed inoculum. The seed is produced by growing the production strain in flasks and smaller fermenters. The volume of the seed inoculum is limited, as a rule of tumb normally 10% of the fermentation volume, to prevent dilution problems. 

The product is extracted from the culture fluid by adsorption onto caibon or resins rather than by solvent. This illustrates an important general point that antibiotic manufacturing processes differ from one another much more in their product recovery stages than in their fermentation stages. Figure 7.4 illustrates a typical production ronte from inoculum to bulk antibiotic. 

For primary isolation of HIV, patient peripheral blood mononuclear cells (PBMC) are collected, the usual inoculum being 106-107 cells. This is the most productive specimen, although virus has been cultured from plasma, semen, tears, saliva, breast milk, and brain tissue. The virus can be cultured from patient specimens at any time in the course of disease, during which the titer changes. Blood contains approximately 60 TCID50% (50% tissue culture infective dose) per milliliter when a person is asymptomatic, and about 7000TCID50/ml in later stages of HIV disease. 

Cultures were incubated with shaking at 250 rpm on a rotary shaker at 37 °C. A 1 % inoculum derived from 8 h stage I cultures was used to initiate fresh LB cultures (200 mL) with antibiotics in a 1 L DeLong flask. These cultures were incubated at 37 °C for 16 h with shaking at 250 rpm. 

Production. The inoculum grew vigorously in the rich yeast extract containing media and produced a thick viscous dispersion in the stirred tank bioreactor. No lignin peroxidase activity could be detected at this stage. When transferred to the 1000 1 production bioreactor, the mycelium of P.chrysosporium attached completely to the nylon wool sheets within a few hours after inoculation and the medium remained completely clear throughout the cultivation. The enzyme had to be harvested immediately after the maximal activity level was reached due to its... 

Estimate the number of generations of growth needed for genetically modified microorganisms from 1 mL culture to a 33,000L production-scale fermenter. Assume that the inoculum size in each stage of the... 

Quantal toxicity tests employing organisms such as daphnids or fish do not alter the concentration of contaminant(s) in a given volume of water because they are directly introduced into their respective experimental containers. In contrast, bioassays undertaken with algae and bacteria somewhat dilute the test material since they must be introduced into test containers (i.e., flasks, tubes or microplate wells) via a certain volume of inoculum. In such tests, the volume share of the test culture can sometimes reach 20 % in the test preparation, which corresponds to a dilution of 1 1.25 (Tab. 2). This dilution stage is therefore the highest concentration that can be examined with such microbial tests. 

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