Mixed Flask Culture

An implicit assumption of the MFC is that the acclimation time is sufficient for coevolution to occur and that coevolution is important to assess the impacts of xenobiotics upon communities. The use of a natural inocula should increase species diversity and complexity over a protocol such as the SAM, but the smaller size of the test vessel would tend to decrease species number. Debate also exists as to the applicability of coevolution in the evaluation of test chemicals. If algal populations and others are primarily regulated by density-independent factors, then population-specific interspecific interactions may not be particularly important. If ecosystems are loosely connected in an ecological sense, coevolved assemblages may be rare. On the other hand, in enclosed systems that are islands, these relationships may have had an opportunity to occur, and coevolved interactions may be important in the assessment of toxicological impacts. 

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Over the last 20 years a variety of multispecies toxicity tests have been developed. These tests, usually referred to as microcosms or mesocosms, range in size from 1 1 (the mixed flask culture) to thousands of liters in the case of the pond mesocosms. A review by Gearing (1989) listed 11 freshwater artificial stream methods, 22 laboratory freshwater microcosms ranging from 0.1 to 8,4001, and 18 outdoor freshwater microcosms ranging from 8 to 18,000,000 1. In order to evaluate and design multispecies toxicity tests, it is crucial to understand the fundamental differences compared to singlespecies tests. A more extensive discussion has been published (Landis, Matthews, and Matthews 1996) and the major points are summarized below. 

Benthic-Pelagic Microcosm Compartmentalized Lake Mixed Flask Culture Microcosm Pond Microcosm Sediment Core Microcosm Ecocore Microcosm Ecocore II Microcosm Standard Aquatic Microcosm Stream Microcosm Waste Treatment Microcosm... 

Summary of Test Conditions for Adaptation of Mixed Flask Culture Microcosms for Testing the Survival and Effects of Introduced Microorganisms... 

Discuss coevolution as a component of the mixed-flask culture microcosm. 

Kim JH, Oh KK, Lee ST, Kim SW, Hong SI (2002) Biodegradation of phenol and chlor-ophenols with defined mixed culture in shake-flasks and a packed-bed reactor. Process Biochem 37(12) 1367-1373... 

Biological. Complete microbial degradation to carbon dioxide was reported under anaerobic conditions by mixed or pure cultures. Under enzymatic conditions formaldehyde was the only product reported (Vogel et al., 1987). In a static-culture-flask screening test, methylene chloride (5 and 10 mg/L) was statically incubated in the dark at 25 °C with yeast extract and settled domestic wastewater inoculum. After 7 d, 100% biodegradation with rapid adaptation was observed (Tabak et al., 1981). 

Biological. Mixed cultures of microorganisms obtained from soil, raw sewage, activated sludge, and settled sludge were all able to degrade azinphos-methyl. When this dithioate pesticide was incubated in a stirred flask containing a mixed culture for 4 d, the concentration decreased from 99 to 40 mg/L (Barik et al., 1984). 

Extraction of the cellulase system. The culture of SSF from each flask (originally 5 g of substrate) was mixed well with more water to bring the final weight of the mixture (mycelium plus unutilized lignin, cellulose, and hemicelluloses) to 100 g. Tween 80 was added at a rate of 0.1%. The mixture was shaken for 0.5 h and centrifuged. The supernatant was used for enzyme determination. We estimated that about 7% to 10% cellulases remained adsorbed on the residues (mycelium and unutilized cellulose, hemicelluloses, and lignin) when the residues were suspended in water and Tween 80 as before and the supernatant was tested for cellulase titer. 

A single colony Is Inoculated Into 100 ml of SeMet media containing antibiotics (Molecular Dimensions). The SeMet media consists of 100 ml SeMet Base, 5 ml nutrient mix, and 0.4 ml 10 mg/ml selenomethionine In a 250 ml shake-flask. The culture Is Incubated at 37°C and 225 rpm overnight ( 16 h). 

The cells in the new flask are immediately mixed to ensure uniform distribution of cells and then cultured at 25°C for 4 d ( eeNote 2). 

Until now, bioreactors of various types have been developed. These include loop-fluidized bed [14], spin filter, continuously stirred turbine, hollow fiber, stirred tank, airlift, rotating drum, and photo bioreactors [1]. Bioreactor modifications include the substitution of a marine impeller in place of a flat-bladed turbine, and the use of a single, large, flat paddle or blade, and a newly designed membrane stirrer for bubble-free aeration [13, 15-18]. Kim et al. [19] developed a hybrid reactor with a cell-lift impeller and a sintered stainless steel sparger for Thalictrum rugosum cell cultures, and cell densities of up to 31 g L1 were obtained by perfusion without any problems with mixing or loss of cell viability the specific berberine productivity was comparable to that in shake flasks. Su and Humphrey [20] conducted a perfusion cultivation in a stirred tank bio-... 

At 13 minutes after initiation of the experiment, transfer 2.5 ml of the culture to a second 125 ml flask containing 0.05 ml of the rifampicin solution prepared in step 3-106. Mix the solution thoroughly. 

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