Physiological state of a microbial community in a biomass recycle reactor

The transition in physiological state was investigated between a carbon-limited chemostat population and microbes growing very slowly in a biomass recycle reactor. The mixed microbial population was metabolizing a mixture of biopolymers and linear alkylbenzene sulfonate, formulated to represent the organic load in graywater. Biomass increased 30-fold during the first 14 days after a shift from chemostat to biomass recycle mode. The ratios of ATP and RNA to cell protein decreased over the first days but then remained constant. The specific rate of CO₂ production by microbes in the reactor decreased 6-fold within 24 h after the shift, and respiratory potentials declined 2–3 fold during the first 7 days. Whereas chemostat cultures used equal proportions of organic carbon substrate for catabolism and anabolism, the proportion of organic substrate oxidized to CO₂ rose from 62 to 82% over the first 8 days in a biomass recycle reactor, and eventually reached 100% as this reactor population exhibited no net growth. Biomass recycle populations removed from the system and subjected to a nutritional shift-up did not immediately initiate exponential growth. The physiological state of cells in the biomass recycle reactor may be distinct from those grown in batch or continuous culture, or from starved cells. Received 02 June 1997/ Accepted in revised form 20 February 1998     

Continuous recombinant bacterial fermentations utilizing selective flocculation and recycle.

Selective recycle has successfully been used to maintain an unstable plasmid-bearing bacterial strain as dominant in a continuous reactor, whereas the culture reverts to 100% segregant cells when selective recycle is not used. The plasmid-bearing strain is slower growing and flocculent; however, when the cells lose their plasmid, the resulting segregant cells are nonflocculent and grow at a faster rate due to their decreased metabolic burden. Both types of cells exit a chemostat and enter an inclined settler where the flocculent plasmid-bearing cells are separated from the nonflocculent segregant cells by differential sedimentation. The underflow from the cell separator, which is enriched with plasmid-bearing cells, is recycled back to the chemostat, while the segregant cells are withdrawn off the top of the settler and discarded. The experimental results agree well with selective recycle reactor theory. On the basis of the theory, a criterion is presented that has been shown to successfully predict whether or not a selective recycle reactor can maintain a plasmid-bearing strain.     

Microbial response to environmental changes in an Anaerobic Baffled Reactor (ABR)

Two 10 litre Anaerobic Baffled Reactors (ABR), with 8 separate compartments, were used to examine the effect of Hydraulic Retention Time (HRT), effluent recycle and temperature changes on the trophic groups in anaerobic digestion. A synthetic carbohydrate (sucrose)-protein substrate was used, and the reactors run at 20 h HRT, and 35 °C. Changing the HRT from 40 to 20 hours doubled the organic loading which caused accumulation of reduced intermediates. The pattern of Volatile Fatty Acids (VFA) at steady state due to an increase in recycle ratios led to the breakage of microbial flocs, and a reduced overall microbial activity. However, the quantity of reduced intermediates was substantially reduced. Decreasing the temperature to 25 °C had differing degrees of influence on reactors I &II, but the same pattern of microbial response occurred; that is the slower growing microorganisms were more affected by the temperature drop. It was found that the unique structure of the ABR brings about the partial separation of acidogenesis and methanogenesis.     

Eco-physiological Characterization of Soil Bacterial Populations in Different States of Growth

The method based on characterization of microbial populations in terms of their growth rate in agar plates has been used for testing the prediction of the theory of r- and K-selection in a microbial community from a tropical soil. Conditions which could lead bacterial populations to grow exponentially or to enter into a stationary phase were obtained by growing soil microbial populations in a chemostat and in a chemostat with recycle, respectively. Significant differences in population distribution patterns were observed by comparing results from the two growth systems. When soil community was grown in a chemostat and subjected specifically to well-defined r- and K-conditions, stable associations of organisms with r- and K-type characteristics developed as a consequence of environmental pressure. In contrast, when cultivated in chemostat with recycle under the same r- and K-conditions imposed on chemostat cultures, distribution patterns of r- and K-selected populations appeared very little affected by changes in substrate availability.     

Pyrosequencing reveals the inhibitory impact of chronic exposure to erythromycin on activated sludge bacterial community structure

The study investigated changes in the microbial population structure sustained at two different sludge ages of 10 d and 2 d under chronic impact of erythromycin. It intended to observe the experimental correlation between variable process kinetics and changes in the composition of the microbial community induced by erythromycin. Samples from two fill/draw reactors operated with continuous erythromycin dosing of 50 mg/L were collected for the analysis of microbial population structure using high-throughput sequencing of 16SrRNA gene. Significant changes were observed in the composition of microbial community during the exposure period. Richness analysis for slower growing culture indicated that most microbial fractions were inactivated and eliminated in favor of fewer more resistant species in different phyla. Sludge age appeared to control the impact of erythromycin on microbial composition. At a sludge age of 2 d, erythromycin appeared to generate richer community with faster growing and more compatible species. For slower growing culture, elimination of vulnerable species was supported by decrease in the number of shared level OTUs. For faster growing culture, shared species level OTUs also decreased significantly upon exposure to erythromycin, suggesting rapid washout and replacement by more resistant species. Resistance gene analysis yielded positive results for mph(A) gene indicating presence of erythromycin-resistant components in the microbial community.     

Manipulation of heterogeneous hybridoma cultures for overproduction of monoclonal antibodies.

In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124) to obtain increased production of monoclonal antibodies (IgG2a), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface-associated antibodies and a fluorescence-activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry.     

Citric acid production by Candida lipolytica Y 1095 in cell recycle and fed-batch fermentors

The effect of dissolved oxygen on citric acid production and oxygen uptake by Candida lipolytica Y 1095 was evaluated in cell recycle and fed-batch fermentation systems. The maximum observed volumetric productivity, which occurred at a dilution rate of 0.06 h(-1), a dissolved oxygen concentration of 80%, and a biomass concentration of 5% w/v, in the cell recycle system, was 1.32 g citric acid/L . h. At these same conditions, the citric acid yield was 0.65 g/g and the specific citric acid productivity was 24.9 mg citric acid/g cell . h. In the cell recycle system, citric acid yields ranged from 0.45 to 0.72 g/g. Both the volumetric and specific citric acid productivities were dependent on the dilution rate and the concentration of dissolved oxygen in the fermentor. Similar productivities (1.29 g citric acid/L . h) were obtained in the fed-batch system operated at a cycle time of 36 h, a dissolved oxygen concentration of 80%, and 60 g total biomass. Citric acid yields in the fed-batch fermentor were consistently lower than those obtained in the cell recycle system and ranged from 0.40 to 0.59 g/g. Although citric acid yields in the fed-batch fermentor were lower than those obtained in the cell recycle system, higher citric:isocitric acid ratios were obtained in the fed-batch fermentor. As in the cell recycle system, both the volumetric and specific citric acid productivities in the fed-batch fermentor were dependent on the cycle time and dissolved oxygen concentration. (c) 1995 John Wiley & Sons, Inc.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

A policy tool for establishing a balance between wildlife habitat preservation and the use of natural resources by rural people in South Africa

In this paper, a model is set up to determine the size of the wildlife population compatible with the extraction of the maximum output by rural people from natural resources. It is found that when the size of human population increases, to obtain the maximum output of food, the size of wildlife population decreases if the human population is growing faster than or at the same rate as that of the wildlife population; whereas the size of the wildlife population increases if the human population is growing slower than that of the wildlife. Furthermore, in the event that the increase in the size of the wildlife population is unable to reach the level compatible with the extraction of the maximum output of food, the improvement of the wildlife habitat and supplementing rural people's income with the proceeds of tourism are proposed as policies to maintain a balance between the preservation of the wildlife habitat and the use of natural resources by rural people.     

Skin fibroblasts from aged Fischer 344 rats undergo similar changes in replicative life span but not immortalization with caloric restriction of donors.

We have compared the in vitro replicative life span and characteristics of immortalization of skin fibroblast cultures derived from ad libitum-fed and caloric-restricted Fischer 344 rats of 6, 24, and 29 months of age. Cells from all 6-, 24-, and 29-month-old animals showed a gradual decline in proliferative potential as evidenced by decreases in harvest density, in the fraction of cells initiating DNA synthesis, and in the number of population doublings per passage. These declines were accompanied by morphological changes including cell enlargement. The replicative life span prior to immortalization decreased significantly with donor age (P less than 0.0001), while caloric restriction had no effect on the cumulative population doubling level. Prior to immortalization mitotic cells from all cultures showed a normal rat karyotype. Postcrisis cultures tended to have more polyploid cells but there were no characteristic or specific chromosomal changes found in the cells with an immortalized phenotype. Interestingly, fibroblasts derived from caloric-restricted animals had a significantly slower growth rate through the tenth week after immortalization (P less than 0.005). When these cultures were seeded at one-quarter the normal seeding density, to favor the outgrowth of the fastest growing cells, a population with a more "transformed" phenotype emerged.     

Methods for determination of growth-rate-dependent changes in hybridoma volume,shape and surface structure during continuous recycle

Hybridoma volume and surface membrane structure were found to vary as a function of specific growth rate using a method of cell recycle with continuous medium perfusion to vary growth rate. Mean hybridoma volume determined at constant osmolality by both electronic particle counting and scanning electron microscopic (SEM) methods indicated that rapidly growing cells are significantly larger than very slowly growing cells. We have previously determined that during both rapid and slow growth over a range of L-glutamine provision rates (Gln PR) that specific monoclonal antibody (MoAb) secretion rate was not changed. In this study a constant MoAb secretion rate per unit of membrane area was found which may indicate that changing membrane area is not a rate-determining factor in MoAb secretion. SEM methods were of limited use for accurate determination of cell volume due to cell shrinkage and large coefficients of variations. In spite of this limitation, SEM stereology methods were useful in confirming that cells remained spherical over a wide range of specific growth rates and that hybridoma cells were not circular. Sequential SEM observations also revealed that surface membrane structure of the 9.2.27 murine hybridoma investigated was correlated with growth rate. Under conditions of very slow growth, hybridoma surface microvilli density appeared to be significantly reduced.     

Response of completely mixed systems to pH shock

The response of aerobically growing heterogeneous microbial populations of sewage origin to step increases and decreases in pH were studied in both once-through and cell recycle systems. The pH range studied was 2.7 to 8.0. All studies were conducted at a dilution rate of 0.125 hr^?1, and all shocks were administered from a base or preshock pH level of 6.4 to 6.7. In each experiment, the preshock or initial “steady state” was assessed, the pH of the feed changed, and the resulting transient behavior of the system examined until attainment of the new or final “steady state” was approached. The major objectives of the work were to characterize the nature of the response with respect to biomass and effluent substrate concentrations, types of microbial populations present and chemical composition of the biomass, and to obtain guidelines as to allowable change in pH in waste streams. It was found in once-through systems that substrate removal efficiency recovered from pH levels as low as 3.0 after rather long periods of transient leakage of substrate. Cell recycle attenuated the severity of substrate leakage. In all cases of severe acid shock, the microbial population changed from predominantly bacterial-protozoan to one consisting predominantly of filamentous fungi. Changes in chemical composition of the sludge (protein and carbohydrate content) were consistent with the population changes. Based upon the results, it can be conservatively estimated that changes in pH of no more than one unit from the neutral preshock range can be tolerated without possible disruption of biochemical efficiency of substrate removal.     

Spherezymes: A novel structured self-immobilisation enzyme technology

Background  

Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts. An alternative is to form self-immobilised enzyme particles.     

Birth, growth and death as structuring operators in bacterial population dynamics

A new model is presented that describes microbial population dynamics that emerge from complex interactions among birth, growth and death as oriented, discrete events. Specifically, birth and death act as structuring operators for individual organisms within the population, which become synchronised as age clusters (called cell generations that are structured in age classes) that are born at the same time and die in concert; a pattern very consistent with recent experimental data that show bacterial group death correlates with temporal population dynamics in chemostats operating at carrying capacity. Although the model only assumes “natural death” (i.e., no death from predation or antimicrobial exposure), it indicates that short-term non-linear dynamic behaviour can exist in a bacterial population growing under longer term pseudo-steady-state conditions (a confined dynamic equilibrium). After summarizing traditional assumptions about bacterial aging, simulations of batch, continuous-flow, and bioreactors with recycle are used to show how population dynamics vary as function of hydraulic retention time, microbial kinetics, substrate level, and other factors that cause differential changes in the distribution of living and dead cells within the system. In summary, we show that population structures induced by birth and death (as discrete and delayed events) intrinsically create a non-linear dynamic system, implying that a true steady state can never exist in growing bacterial populations. This conclusion is discussed within the context of process stability in biotechnology.     

The population dynamics of Metapenueus ensis (Crustacea: Decapoda: Penaeidae) in a traditional tidal shrimp pond at the Mai Po Marshes Nature Reserve, Hong Kong

The population dynamics of Metapenaeus ensis in a traditional tidal shrimp pond (locally known as gei wai ) at the Mai Po Marshes Nature Reserve, Hang Kong, were studied over a 29-month period. The population of M. ensis was dominated by either one or two age groups throughout the year. Life span was < 16 months. M. ensis is sexually dimorphic in terms of size, with females growing faster and attaining a larger maximum size than males. The sex ratio did not differ significantly from 1:1. The prawns do not mature and reproduce in the gei wai so that the population is sustained by postlarval immigration which begins in June and ends in December, with peaks in August and November. The gei wai population of M. ensis is characterized by slower growth, lower maximum attainable size, shorter life span, reproductive sterility and an incomplete life cycle, as compared with 'natural' population in the coastal waters of Hong Kong.     

Connexins in wound healing; perspectives in diabetic patients

Skin lesions are common events and we have evolved to rapidly heal them in order to maintain homeostasis and prevent infection and sepsis. Most acute wounds heal without issue, but as we get older our bodies become compromised by poor blood circulation and conditions such as diabetes, leading to slower healing. This can result in stalled or hard-to-heal chronic wounds. Currently about 2% of the Western population develop a chronic wound and this figure will rise as the population ages and diabetes becomes more prevalent [1]. Patient morbidity and quality of life are profoundly altered by chronic wounds [2]. Unfortunately a significant proportion of these chronic wounds fail to respond to conventional treatment and can result in amputation of the lower limb. Life quality and expectancy following amputation is severely reduced. These hard to heal wounds also represent a growing economic burden on Western society with published estimates of costs to healthcare services in the region of $25B annually [3]. There exists a growing need for specific and effective therapeutic agents to improve healing in these wounds. In recent years the gap junction protein Cx43 has been shown to play a pivotal role early on in the acute wound healing process at a number of different levels [4-7]. Conversely, abnormal expression of Cx43 in wound edge keratinocytes was shown to underlie the poor rate of healing in diabetic rats, and targeting its expression with an antisense gel restored normal healing rates [8]. The presence of Cx43 in the wound edge keratinocytes of human chronic wounds has also been reported [9]. Abnormal Cx43 biology may underlie the poor healing of human chronic wounds and be amenable therapeutic intervention [7]. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Effect of growth in highly salinized media on the enzymes of the photosynthetic apparatus in pea seedlings

The rate of chlorophyll formation in initially etiolated pea seedlings (Pisum sativum) that are growing in the light in salinized media is slower than in similar plants not subjected to salinity. However, the final steady state level of chlorophyll is the same under both conditions. Growth under saline conditions did not change the ratio of dry weight to wet weight in the plant leaves nor the specific concentration of soluble protein in leaf extracts. Changes in the specific activity of 11 enzymes in leaf extracts during growth in the light were measured. At least six of these enzymes are known to be part of the photosynthetic apparatus and that their synthesis is subject to photocontrol. The changes in specific activity that were observed were slower in the salt-treated plants, but the final steady state concentration of each was the same as in the control plants. It is concluded that salinity impairs growth of pea plants but that formation of enzymes and other proteins are always in balance with growth.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Variation in a natural population of Schizophyllum commune. Variation within the extreme isolates for growth rate.

Two extreme dikaryotic idolates chosen from a large sample of a localised population of Schizophyllum commune exhibited a considerable amount of genetical variation for growth rate at the near ambient temperature of 20 degrees C and at the higher temperature of 30 degrees C. The potential variation within these extreme isolates were greater than the variation observed in the whole sample. Regression analysis of the variation in growth rate of the dikaryotic progeny of the extreme isolates on that of their component monokaryons showed that the nature of gene action was not the same in these two stages of the life cycle. The simple additive-dominance model of gene action was adequate to explain the variation in growth rate in both of the extreme isolates at both of the temperatures. The small deviations from this model could be accounted for by unequal gene frequencies due to small sample size although a low incidence of non-allelic interactions could not be rule out. Directional dominance for growth rate was detected in both isolates at the more normal temperature and it was opposing in direction in the two isolates. In the slow growing isolate the dominance was for faster growth and in the fast growing isolate it was for slower growth. This is expected for a character which displays overall ambi-directional dominance if isolates with more extreme growth rates than those recovered in the population sample are eliminated by stabilising selection. The dominance is temperature dependent being ambi-directional in both isolates at the higher temperature. Environmental heterogeneity, the buffering effects of directional dominance and genotype-environment interactions and opposing selective forces operating on the monokaryotic and dikaryotic stages of the life cycle are possible contributory factors to the considerable free and potential variability displayed in this small, localised population.