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Mammalian cell culture has obtained high relevance for the production of recombinant therapeutic and diagnostic proteins from established cell lines. Furthermore, great effort has also been made to grow primary and undifferentiated cells in vitro. In each case it is of great importance for the establishment of a certain culture to know the demands of the cells as exactly as possible. Apart from global parameters like temperature, pH, oxygen tension, and osmolarity, the availability of substrates and the concentration of metabolic waste products play a major role in the performance of the cell culture. In order to monitor the concentrations of these substances and to evaluate the state of the culture, some different methods have to be applied. There is a broad spectrum of well-defined procedures available to estimate the concentration of each of the important substances. In general, it is very much recommended to use standardized working instructions and automatic systems to obtain trustworthy analysis results. Some such reliable methods are listed in this chapter. These procedures are all established in our laboratory and employed routinely during daily work. They should be easily transferred and installed in another lab.

The enormous growth of industrialization and agriculture has resulted in serious environmental pollution, and polychlorophenols are among the most hazardous pollutants. Because of the large investment required for traditional physical and chemical detoxification methods, engineering transgenic plants for phytoremediaton of polluted soil has received more and more attention. In most cases, however, the plants are employed to take up the pollutants and the toxic compounds are often not destroyed but merely accumulated or displaced. Recently, we developed a novel system of phytoremediation ex planta based on the overexpression of a secretory laccase that catalyzes the oxidation of various aromatic compounds, including 2,4,6-trichlorophenol. Because there are rich sources of various detoxifying enzymes, the technique of using plant exudation machinery for phytoremediation should be applicable to other types of pollutants in soil.

Studies on plant-mediated decontamination of polluted waters commonly rely on hydroponic bioreactor systems for the rapid screening of different plant species and the investigation of the phytoremediation mechanisms. This chapter describes the use of ebband-flow and sealed root-zone hydroponic bioreactors (some mounted with dissolved oxygen, pH, and E_H probes) to investigate, under aerobic and anaerobic rhizosphere conditions, respectively, the phytoremediation potential of the new and emergent contaminant perchlorate. The experimental data show slower kinetics and higher fraction uptake and phytodegradation of perchlorate by plants grown under predominantly aerobic root-zone conditions. Meanwhile, for plants grown in sealed (predominantly anaerobic) root-zone hydroponic systems, the bulk solution chemistry is influenced by the plant photosynthesis and respiration cycles, and both phytodegradation and rhizodegradation are the predominant phytoprocesses. In ongoing research, various approaches are being developed to minimize the undesired uptake and slow phytodegradation process and enhance the favorable and rapid rhizodegradation process. Additionally, planted soil bioreactor experiments that simulate phytoremediation of perchlorate under natural field conditions are described.

In India, urbanization, excessive utilization of natural resources, and population growth are the causes for air, water, and soil contamination and pollution. Major environmental problems in India are land degradation (deforestation, overgrazing, overcultivation, faulty irrigation), destruction of wildlife habitat and erosion of genetic resources (including those of crops and trees, terrestrial animals, and fish), and pollution (air, water, and soil pollution with toxic wastes and other substances). Soil conservation and restoration of degraded soils (wasteland/marginal land) is the most serious environmental concern to India. In India, soil erosion is a serious problem ranging from loss of top soil in 130.5 million ha to terrain deformation in 16.4 million ha. Soil loss under different land-use options has been reported and minimum loss found when trees and grass were grown together in a silvipastoral system. For e.g., Shivaliks (foothills of Himalayas, one of the most fragile ecosystems) has included combinations of eucalyptus-bhabar grass; Acacia catechu-forage grass; Leucaena-Napier grass; teak-Leucaena-Bhabar; Eucalyptus-Leucaena-Turmeric; poplar-Leucaena-Bhabar; and Sesamum-rape seed. Sodic soils of the Indo-gangetic alluvial plain are characterized by high pH, high exchangeable sodium and phosphorus, low infiltration, dispersed soil, low organic matter content, and poor fertility. Special planting techniques have been developed for raising multipurpose tree species in sodic and saline soils. A silvipastoral model comprising Prosopis juliflora and Leptochloa fusca has been developed, and alkali soils have been standardized. Another serious problem is the physical deterioration of soil because of water logging or submergence/flooding that has affected around 11.6 million ha of land in India. Suitable trees and grass species for such situations are trees (Eucalyptus tereticornis, Populus deltoids, Terminalia arjuna, Acacia auriculiformis, Syzigium cumini, Albizia lebbek, Dalbergia sissoo, and Pongamia pinnata) and grasses (para grass, cord grass, lemon grass, and Setaria grass). Contamination of food and other agricultural products with pesticide residues is a widespread problem in India. India’s 15 oil refineries generate a huge amount of oily sludge annually. This also takes a toll on the scarce soil, because land requirements increase with an increase in oil sludge generation. Besides the sludge from oil refineries, crude oil spills too are a cause of environmental degradation. The “Mission Mode” experiment of fly-ash management including using fly ash in forestry systems is one of the important strategies to protect environmental degradation.

MAbs (mAbs) are unique and versatile molecules that have found applications in research, in vitro and in vivo diagnostics, and the treatment of several diseases. Antibody technology has been revolutionized by the hybridoma technology for rnAb production described by Köhler and Milstein in 1975 (1). In the last 30 yr a number of genetically engineered antibody constructions have emerged, including chimeric and human-like antibodies as well as different antibody fragments. Nowadays, 18 rnAb products are currently on the market and more than 100 are in use in clinical trials (2). The total market for biopharmaceuticals like therapeutic mAbs is a multibillion dollar opportunity, with cancer and arthritis as the major therapeutic applications, with estimated total markets of $15 and $25 billion in 2010, respectively (3). In this chapter we give an overview of the state of the art of antibody engineering and production methods and describe standard protocols for producing a hybridoma cell line, the building up of transfectomas, and a suitable bacterial expression system for the production of antibodies and antibody fragments established in our laboratory.

Metabolic flux analysis with its ability to quantify cellular metabolism is an attractive tool for accelerating cell line selection, medium optimization, and other bioprocess-development activities. In the stoichiometric flux estimation approach, unknown fluxes are determined using intracellular metabolite mass balance expressions and measured extracellular rates. The simplicity of the stoichiometric approach extends its application to most cell culture systems, and the steps involved in metabolic flux estimation by the stoichiometric method are presented in detail in this chapter. Specifically, overdetermined systems are analyzed because the extra measurements can be used to check for gross measurement errors and system consistency. Cell-specific rates comprise the input data for flux estimation, and the logistic modeling approach is described for robust specific rate estimation in batch and fed-batch systems. A simplified network of mammalian cell metabolism is used to illustrate the flux estimation procedure, and the steps leading up to the consistency index determination are presented. If gross measurement errors are detected, a technique for determining the source of gross measurement error is also described. A computer program that performs most of the calculations described in this chapter is presented and references to flux estimation software are provided. The procedure presented in this chapter should enable rapid metabolic flux estimation in any mammalian cell bioreaction network by the stoichiometric approach.

This chapter describes the latest ultrasonic nebulizer technology for the production of inocula of arbuscular mycorrhizal fungi for the purpose of mycorrhizo-remediation of metal-contaminated soils. It is a superior alternative to common pot-culture and conventional atomizing disc or spray nozzle systems used for the production of arbuscular mycorrhizal fungi inocula. This technology employs high-frequency sound to nebulize nutrient solution into microdroplets 1 μm in diameter. Fast growth of roots of the test plant, precolonized with arbuscular mycorrhizal fungus obtained from the contaminated site, is achieved in the ultrasonic nebulizer chamber containing low-P nutrient solution. These roots can be sheared and used as inoculum in small doses to improve survival and growth of plants used for mycorrhizo-remediation of metal-contaminated soils, which is a prerequisite for phytoremediation.

Encapsulation of cell cultures is a universally applicable tool in cell culture technology. Basically it can be used to protect cells against hazardous environmental conditions as well as the environment against disadvantageous effects triggered by the immobilized cultures. The three basic encapsulation systems, described here, are the bead, the coated bead, and the membrane-coated hollow sphere. Another two universally applicable methods to quantify the number of living cells immobilized in those encapsulation systems could be used.

Fixed-bed reactors for cultivation of mammalian cells can be used for a variety of applications such as the production of monoclonal antibodies with hybridoma cells or proteins for diagnostic or therapeutic use with recombinant, mostly anchorage-dependent animal cells, production of retroviral vectors, as well as for cultivation of tissue cells for artificial organs. In Subheading 2., suppliers for fixed-bed reactor systems and macroporous carriers as well as components of a lab-scale fixed-bed reactor and important mass balance equations are presented. Subheading 3., covers topics such as selection of suitable carriers, procedures for cultivation in lab-scale fixed-bed reactors (setting up of the reactor, autoclaving, sterility test, preparation of seed culture and inoculation, start-up and process monitoring during culture, batch and continuous operation, activities after the culture), and additional requirements for larger reactor volumes (setup and sterilization, “inoculation during cultivation strategy”).

The development of flow cytometric techniques has greatly advanced our ability to identify and characterize the morphological and biochemical heterogeneity of cell populations, permitting a rapid and sensitive cell analysis. In this chapter, various flow cytometric applications in cell culture are described for cell lines grown on a laboratory scale, but the principles can also be employed for the monitoring of cell lines up to the large industrial scale.