CO +)O + AH. CO. + H,O A (2) NH3 4-OA + AH-NHOH + H.O + A (3) T'he immediate source of reductant (AH.) can be reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in methanotr-ophs, but in ammonia oxidizeirs it may be a cytochroome c (25. 157). All of the above oxidations are pparently catalyzed by monooxygenase enzymes: methane monooxygenase in methanotrophs and ammonia monooxygenase in ammonia oxidizei-s (25, 61). Only CH4 can support growth in the former organisms. and only NH. can support growth in the latter (39. 79. 80, 139). In both methanotrophs and ammonia oxidizers, there are reports that CH,OH is metabolized to CO, and cell C (25, 79, 164). While the pathway of CH,OH oxidation is well characterized in methanotrophs, in ammonia oxidizers it remains incompletely understood (148, 149). In addition, during the production of NO. from NH4' by both methanotrophs and ammonia oxidizers, small amounts of N,O are evolved (174, 177, 178). Although there is evidence that, in certain environments, methanotrophs and armmonia oxidizers may carry out oxidation of the growth substrate of the other as well as CO, few, if any, attempts have been made to measure directly the 68

Growth, nutrient consumption, and end-product accumulation were quantitated in shake-flask cultures of two insect cell lines, Sf-9 and BTI-EAA, in three different serum-supplemented media. Per cell consumption or production rates were calculated for most medium components analyzed. Glucose was growth-limiting in TNM-FH medium and was the most important single source of organic-C for the cells in all cultures. Cells utilized fructose and maltose but not sucrose. alpha-Ketoglutarate and malate contributed significantly to the carbon budget of cells in TNM-FH. Lactate generally did not accumulate during growth. Most of the amino acids were consumed by the cells, with the exception of alanine which was produced. Most of the amino acids appeared to be present in adequate supply in the cultures. Glutamate was generally the most rapidly consumed of the amino acids, followed closely by glutamine. Alanine accumulation was correlated with glucose consumption. In Sf-9 cultures, ammonia accumulated only slightly or not at all as long as glucose was present in the medium, and uric acid was detectable at the end of growth and in the stationary phase. Added ammonia up to a concentration of 10 mM did not affect the growth of either cell line. Ammonia and lactate may be of less importance in limiting growth in insect cell cultures than in mammalian cell cultures. A hypothetical outline of the major metabolic pathways of the cultured insect cells is presented on the basis of information obtained here and in the literature.

Ammonia oxidizers (family Nitrobacteraceae) and methanotrophs (family Methylococcaceae) oxidize CO and CH4 to CO2 and NH4+ to NO2-. However, the relative contributions of the two groups of organisms to the metabolism of CO, CH4, and NH4+ in various environments are not known. In the ammonia oxidizers, ammonia monooxygenase, the enzyme responsible for the conversion of NH4+ to NH2OH, also catalyzes the oxidation of CH4 to CH3OH. Ammonia monooxygenase also mediates the transformation of CH3OH to CO2 and cell carbon, but the pathway by which this is done is not known. At least one species of ammonia oxidizer, Nitrosococcus oceanus, exhibits a Km for CH4 oxidation similar to that of methanotrophs. However, the highest rate of CH4 oxidation recorded in an ammonia oxidizer is still five times lower than rates in methanotrophs, and ammonia oxidizers are apparently unable to grow on CH4. Methanotrophs oxidize NH4+ to NH2OH via methane monooxygenase and NH4+ to NH2OH via methane monooxygenase and NH2OH to NO2- via an NH2OH oxidase which may resemble the enzyme found in ammonia oxidizers. Maximum rates of NH4+ oxidation are considerably lower than in ammonia oxidizers, and the affinity for NH4+ is generally lower than in ammonia oxidizers. NH4+ does not apparently support growth in methanotrophs. Both ammonia monooxygenase and methane monooxygenase oxidize CO to CO2, but CO cannot support growth in either ammonia oxidizers or methanotrophs. These organisms have affinities for CO which are comparable to those for their growth substrates and often higher than those in carboxydobacteria. The methane monooxygenases of methanotrophs exist in two forms: a soluble form and a particulate form. The soluble form is well characterized and appears unrelated to the particulate. Ammonia monooxygenase and the particulate methane monooxygenase share a number of similarities. Both enzymes contain copper and are membrane bound. They oxidize a variety of inorganic and organic compounds, and their inhibitor profiles are similar. Inhibitors thought to be specific to ammonia oxidizers have been used in environmental studies of nitrification. However, almost all of the numerous compounds found to inhibit ammonia oxidizers also inhibit methanotrophs, and most of the inhibitors act upon the monooxygenases. Many probably exert their effect by chelating copper, which is essential to the proper functioning of some monooxygenases. The lack of inhibitors specific for one or the other of the two groups of bacteria hampers the determination of their relative roles in nature.

Significant improvement in cell growth and protein production has been achieved in Sf‐9 insect cell cultures using pulse additions of multicomponent nutrient feed concentrates (Bédard et al., 1994; Chan et al., 1998). The present work focuses on investigating an alternative feeding strategy wherein the nutrients are fed in a semi continuous manner. Fed batch culture experiments were carried out to compare the two different feeding strategies, pulse and semi continuous and a process developed to achieve a cell density of 5.2 × 107 cells/mL of Sf‐9 cells in a 3.5 L bioreactor. Production of recombinant protein β‐galactosidase was carried out by infecting the cells with baculovirus at a MOI of 10 at cell densities of 17 × 106cells/mL. Specific productivity could be maintained at cell densities as high as 14 × 106 cells/mL. The results presented indicate that the feeding method can provide significant improvements in the performance with a reduction in the amount of total nutrients added. On‐line monitoring of the culture using the capacitance probe showed that the capacitance probe can be used successfully to monitor the biomass and infection process even at higher cell densities. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 381–388, 2000.