Biomass indirect measures

The Microplate algal toxicity test, another popular small-scale bioassay, is equally employed for WASTOXHAS applications (see Chapter 3, volume 1 of this book). In our case, rapid endpoint determinations of growth (72h-IC50s) are made with a microplate fluorescence reader, where algal biomass is indirectly measured via chlorophyll a fluorescence (excitation filter 440 nm - emission filter 640 nm). 

The first persons to point out the possibility of computer monitoring of indirectly measured parameters were Yamashito, Hisashi, and Inagaki in 1969 (2). The method was described in a U.S. Patent in 1975 (3). Examples of the application of computer-aided indirectly measured parameters to the control and optimization of batch-fed Baker s Yeast fermentation were described by Jefferis and Humphrey in 1973 (4) and by Wang, Cooney, and Wang in 1977 (5). Background and detailed history of this application can be found in the review by Humphrey (1). The work to be discussed in this report is another example of computer-aided indirectly measured cell biomass and growth rate and the use of this information in the feed back control of the carbon substrate, ethanol, in a process for the production of a yeast single cell protein (SCP). 

Indirect Measurement of Cell Biomass and Growth Rate... 

The possibility of indirectly measuring cell biomass and growth rate by computer-aided on-line substrate balancing was first mentioned in a publication by Humphrey (11). The method is discussed in detail in a review by Humphrey (1). The technique operates by noting that the difference in amount of a particular component in and out of the vessel is equal to that used by the organism for growth and maintenance if no appreciable secondary metabolites are formed. Mathematically this can be stated as... 

The latter requirement and the concomitant improvement of comfort led to the exploitation of alternative methods to estimate the biomass concentration. All of them have in common (1) that they are indirect measures, and (2) that models are mandatory to relate these measures to biomass concentration. The models are of the descriptive rather than the mechanistic type which means... 

Fig. 6 Correlation between the lipase activity and the biomass contents indirectly measured by the glycosamine content and the digital image processing...

State, types of functional groups), redox potential, pH, nutrient and carbon availability, contaminant bioavailability and concentration, electron acceptors, temperature, salinity, and microbial consortia and biomass (D Angelo, 2002). Reaction rates can vary over several orders of magnitude depending on these environmental factors. Studies have documented the effects of several of these factors on rates of mineralization of contaminants in wetland substrates. Redox potential, a measure of the electron availability and an indirect measure of the oxygen status, has been used to show certain compounds degrade favorably under aerobic conditions (e.g., naphthalene), others under anaerobic conditions (e.g., DDT), and still others under moderately anaerobic conditions (e.g., polychlorobi-phenyls [PCBs]). 

The Rothamsted Carbon Model (RothC) uses a five pool structure, decomposable plant material (DPM), resistant plant materials (RPM), microbial biomass, humified organic matter, and inert organic matter to assess carbon turnover (Coleman and Jenkinson 1996 Guo et al. 2007). The first four pools decompose by first-order kinetics. The decay rate constants are modified by temperature, soil moisture, and indirectly by clay content. RothC does not include a plant growth sub-module, and therefore NHC inputs must be known, estimated, or calculated by inverse modeling. Skjemstad et al. (2004) tested an approach for populating the different pools based on measured values. 

A final optical application deals with the measurement of intracellular nicotinamide adenine dinucleotide (NADH) by fluorescence [77], giving information about the physiological status of wastewater treatment plant biomass. This indirect method could be envisaged for toxicity estimation. 

The measurements of the labeled metabolites may be performed with GC- or LC-MS, or by NMR. Because it is the most commonly used method, we will only consider GC-MS based approaches here. Obviously and unfortunately, it is not possible to directly measure the isotopomer enrichments by GC-MS, because the apparatus only yields total masses of molecules or fractions thereof, but not directly the position of a label. Each MS peak is produced by all isotopomers with the same molecular weight that is, the same number of labeled carbon positions. Sometimes this concept is also called mass isotopomers [264]. In a so-called retrobiosynthetic approach, it has been shown that the labeling state of many intracellular pools can be determined indirectly by measuring the labels in macromolecular biomass components at steady state for example, the labeling state of alanine from hydrolyzed protein reflects the label of pyruvate [265]. Using this approach, it is possible to quantify fluxes into storage components. 

The determination of the biomass concentration in complex media by weight is not possible. Therefore, indirect methods were tested. The OTR and CPR are not suitable for the biomass evaluation, because RQ considerably varies during the cultivation. In the first 60 h, it increases, between 60 and 80 h it drops sharply and only after 100 h does it attain a constant value. However, after 100 h, the biomass concentration can be measured by weight in any case. The best agreements were obtained between RNA and biomass concentrations in semisynthetic media. The biomass concentration increases with cultivation time up to 140 h. 

Measurement of soil activity there are a number of laboratory methods which are suitable for measuring the biological activity of the soil. In principle, a distinction is made between direct and indirect methods for the determination of soil activity. The biomass in the soil, for example, can be estimated by counting the individual organisms in the soil, or the measurement of respiration after the addition of a nutrient in excess can provide an indication of active biomass. Moreover, in determinations of activity, a distinction is made between actual and potential activity. Actual activity values are values measured at the time that the sample was taken. Determinations of potential activity, on the other hand, show the level of performance that microorganisms are capable of under optimum experimental conditions, after the addition of a nutrient substrate and prolonged incubation. 

Indirect methods, based on allometric inference, from measurements of the diameter at breast height (DBH) and the height of the trees to obtain wood volumes, is the main method adopted to estimate biomass in the Amazon. Forest biomass estimates are made through regression analysis, where several fitting curves are tested to obtain an ideal model that can be applied to the trees. These models are calibrated by direct weighing of the biomass from a subsample of trees (see for example, Jordan and Uhl 1978, Higuchi et al. 1994, Brown et al. 1995), and could also include other compartments besides trees, such as vines or understory. Indirect methods are broadly adopted in the forestry industry to evaluate the volume of commercial wood. 

Trophic state can be measured directly via indicators of phytoplankton biomass and nutrient concentrations or indirectly by measuring water transparency (Table 1). Water transparency can be an accurate indicator of phytoplankton productivity, even though it is affected by nonbio-logical particles suspended in the water column. Secchi disk depth is the oldest, simplest, and quickest quantitative measure of water clarity. A Secchi disk is a black and white disk that is... 

Davis et al (1985) screened 57 dif rent woody and herbaceous biomass feedstocks for their production of liquid hiel constituents from indirect liquefaction of powdered materials in a fluidized b. Composition metrics in Davis studies were ash, proteins, polyphenols, oil, and hydrocarbon content as defined and measured by the USOA (Ibr example, in Adams, et al, 1986 Buchanan, et al, 1980a Roth et al, 1984 Swanson, et al 1979). We will call the USDA-measured compositions traditional" fcr purposes of this article. These feed compositions were correlated with pyrolysis gas production of hydrogen, CO, CjH4, total olefins, paraffins, and H2/CO ratio using direct regression equations of the quadratic type, but tar/char production was not addressed. 

Second, interpretive models can be proposed. These models take as input operating variables of the bioreactor and measurements of those state variables which it is practical to measure, and give as output estimates of other state variables, including state variables which it may be impossible or impractical to measure during the fermentation. Such models are quite useful in SSF, because it is not practical to obtain direct measurements of the biomass, and parameters which can be measured on-line such as oxygen or carbon dioxide concentrations are only indirectly related to the amount of biomass. The accuracy of such models can be checked by predicting state variables which are easily measured experimentally, such as bed temperature. These models are interpretive and not predictive because they rely on the constant input of fermentation data they cannot predict bioreactor performance simply on the basis of initial conditions. However, they are still quite useful since, if the measured variables can be measured on-line, the model can be used quite successfully in control schemes. 

Off-line measurements of biomass are also problematic because it is usually impossible to separate the fimgal biomass from the substrate. Therefore indirect methods of following growth have to be used. A wide range of methods can be used, but most fall into three cat ories - direct separation of the biomass from the solid matrix, measuring metaboUc activities, and measuring biomass components. 

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