Metabolic shift analysis at high cell densities

Abstract: In high cell density cultures it is virtually inevitable that the environment to which the cells are exposed is heterogeneous. Thus, with suspended cultures, individual cells are subject to temporal changes in their environment whereas with aggregated or immobilized cells, the culture can be considered as being formed by a number of subpopulations, each with its own environmental characteristics. In addition, in a high cell density environment, high concentrations of end products may negatively influence the growth rate. This may result in the selection of organisms with an altered metabolic behaviour or with a decreased sensitivity to the adverse effects of the product. We discuss the consequences of this heterogeneity with regard to carbon source metabolism in view of the ability of many bacterial species to adapt to environmental conditions. Selection of variant organisms was found to occur with Clostridium butyricum when grown for a prolonged time in a medium containing approx. I-50 mM glucose. In contrast to the original strain, these variants could sustain a high maximal growth rate in the presence of butyric acid. In addition, they had acquired the capacity to spontaneously form aggregates and were able to carry out a completely solventogenic fermentation. Heterogeneous metabolic activity in aggregated cells is demonstrated with cultures of Lactobacillus laevolacticus , an aggregateforming lactic acid bacterium that converts glucose completely to o-lactate. By using microelectrodes, we show that the fraction of metabolically active cells decreases with increasing aggregate size: in larger aggregates steep pH gradients occur with the effect that only the outer layer of the aggregate is metabolically active, i.e. contributes to lactic acid formation, whereas with smaller aggregates all cells remain active. As a result, the net specific lactic acid production rate of the population as a whole is not invariably increased with increased aggregate size.     

Patterns of short-term amino acid accumulation and loss in the root-zone of liquid-cultured forage rape (Brassica napus L.)

Amino acid release from roots of sterile and non-sterile, solution-grown, 7-, 21- and 60-days-old forage rape plants (Brassica napus L.), was measured over periods of up to 6 hours. With sterile plants, release of amino acids into a fixed volume of collection medium (6, 12, 70 mL) was concentration-limited, giving rise to similar convex accumulation profiles for individual acids. In contrast, amino acid accumulation in continuously circulating collection medium was not concentration limited, giving a linear accumulation pattern. The compositions of accumulating amino acids, which were similar to those measured in root extracts, did not change significantly. However, the proportions of ALA, GABA, GLU and ILE in both root extracts and root-derived amino acids increased as plants aged. Older plants released more amino acids per plant, while younger plants released more amino acids g^-1 root DW. Using non-sterile plants, the patterns of change in amino acid concentration and composition in the collection medium were completely different from those determined with sterile plants. In general, with 7-days-old plants, and 60-days-old plants that had recently become non-sterile, an initial rise in the concentration of all acids was followed by a fall to low levels. The loss of amino acids was apparently due to microbial consumption. Individual amino acids attained maximum concentration at different times during the collection process. This is attributed mainly to concentration-dependent differential assimilation of amino acids, since those with the highest initial concentrations, the major components of the mixtures released from roots, declined the earliest. When calculated rates of amino acid release from roots (R_r) and microbial consumption of amino acids (R_c) were compared (for 7-days-old plants), the highest ratios of R_c/R_r were found for ASN, ARG, GLU, GLN, and LYS. This suggests a degree of selectivity for glutamate and nitrogen-rich acids on the part of the consuming micro-organisms. With 21-days old plants and 60-days old plants grown entirely under non-sterile conditions, fluctuations in amino acid concentration were similar for all acids.     

Changes in populations of soil microorganisms,nematodes, and enzyme activity associated with application of powdered pine bark

Evaluation of enzyme activities in combination with taxonomic analyses may help define the mechanisms involved in microbial decomposition of orgaic amendments and biological control of soilborne pathogens. In this study, powdered pine bark was added to nematode-infested soil at rates of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 g kg^–1. Total fungal populations did not differ among treatments immediately after application of pine bark. After 7 days, fungal populations were positively correlated with increasing levels of pine bark. This increase was sustained through 14 and 21 days.Penicillium chrysogenum andPaecilomves variotii were the predominant fungal species isolated from soil amended with pine bark. Total bacterial populations did not change with addition of pine bark at 0, 7, and 14 days after treatment. At 21 and 63 days, total bacterial populations declined in soil receiving the highest rates of pine bark. Addition of pine bark powder to soil caused a shift in predominant bacterial genera fromBacillus spp. in nonamended soil, toPseudomonas spp. in amended soil. Soil enzyme activities were positively correlated with pine bark rate at all sampling times. Trehalase activity was positively correlated with total fungal populations and with predominant fungal species, but was not related to bacterial populations. The number of non-parasitic (non-stylet bearing) nematodes andMeloidogyne arenaria in soil and roots were not correlated with pine bark rate. However,Heterodera glycines juveniles in roots, and the number of cysts g^–1 root, declined with increasing levels of pine bark.Journal Series Series No. 18-933598 Alabama Agricultural Experiment Station     

Microbial indicators for sawdust and bark compost stability and humification processes

To what extent some microbial index ratios are suitable for use as early criteria for the level of compost stability during aerobic composting of coniferous sawdust and bark at mesophilic conditions was studied. Evolution of the specific respiration activity (CO₂-C/biomass C) and the ratios between some groups of microorganisms were followed as a function of composting time. The specific respiration activity was found to be an early and most reliable indicator of compost stability. The peroxidase and polyphenoloxidase enzyme activity during composting, as well as the composition of newly-formed humus substances were studied. The duration of composting increased the quality of newly-formed humus substances (C_h.a.:C_f.a ratio; Ca-complexed humic acid and resistance of organo-mineral complexes). The quality of humus substances could be used to assess compost stability. However, the results can be applied only under defined conditions.     

The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.     

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.     

Impalement of marine turtles (Reptitia,Chelonia: Cheloniidae and Dermochelyidae) by billfishes (Osteichthyes,Perciformes: Istiophoridae and Xiphiidae)

Synopsis Billfishes have long been known to impale a great variety of objects, but there are only two brief, obscure records of marine turtles being speared. Details are presented on these two, as well as on two other confirmed records; data from two additional unconfirmed records are also presented. In total, three species of marine turtles are known to have been impaled by three species of billfishes; a fourth species of fish and a fourth species turtle are listed in an unconfirmed case. Records come from the eastern and western Pacific as well as the eastern Atlantic. Of the four confirmed cases, the turtles survived in two, and apparently died as an effect of the spearing in the other two. In three confirmed cases only the impaled rostrum was encountered, and in one confirmed case the entire fish was found, with its rostrum piercing the turtle. There is no obvious advantage — or clear disadvantage — involved in impaling turtles. It is argued that these attacks are accidental, and the result of attempts made by the billfish to capture prey that are near the turtle. These spearings indicate that the chelonians serve as shelters for prey animals on the high seas, and thus, are further evidence of the pelagic existence of marine turtles. The impalings are evidence of a singular ecological role of the turtles — as live fish aggregation devices.     

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

Algae as indicators of environmental change

Despite an increased awareness by governments and the general public of the need for protecting all types of aquatic habitats, human impacts continue to impair the services that these ecosystems provide. Increased monitoring activities that locus on all major biological compartments are needed to quantify the present condition of Earth's aquatic resources and to evaluate the effectiveness of regulations designed to rehabilitate damaged ecosystems. Algae are an ecologically important group in most aquatic ecosystems but are often ignored as indicators of aquatic ecosystem change. We attribute this situation both to an underappreciation of the utility of algal indicators among non-phycologists and to a lack of standardized methods for monitoring with algae.Because of their nutritional needs and their position at the base of aquatic foodwebs, algal indicators provide relatively unique information concerning ecosystem condition compared with commonly used animal indicators. Algae respond rapidly and predictably to a wide range of pollutants and, thus, provide potentially useful early warning signals of deteriorating conditions and the possible causes. Algal assemblages provide one of the few benchmarks for establishing historical water quality conditions and for characterizing the minimally impacted biological condition of many disturbed ecosystems. Preliminary comparisons suggest that algal indicators are a cost-effective monitoring tool as well.Based on available evidence from field studies, we recommend development of taxonomic indicators based on diatoms (Bacillariophyceae) as a standardized protocol for monitoring ecosystem change. Both population- and community-level indices have inherent strengths, and limitations and information from both levels of biological organization should be utilized in tandem. However, further information concerning species tolerances to a variety of anthropogenic stressors is needed if autecological indices are to be used routinely for monitoring purposes. While functional measures (e.g. productivity) may also prove useful as monitoring tools, further investigation is required to characterize the reliability of alternative methodologies and to assess the consistency of these indicators under varying field conditions.Author for correspondence