Antarctic sea ice viral dynamics over an annual cycle

Viral abundance, burst sizes, lytic production and temperate phage were investigated in land-fast ice at two sites in Prydz Bay Antarctica (68°S, 77°E) between April and November 2008. Both ice cores and brine were collected. There was no seasonal pattern in viral or bacterial numbers. Across the two sites virus abundances ranged between 0.5 × 10⁵ and 5.1 × 10⁵ viruses ml⁻¹ in melted ice cores and 0.6 × 10⁵–3.5 × 10⁵ viruses ml⁻¹ in brine, and bacterial abundances between 2.7 × 10⁴ and 17.3 × 10⁴ cells ml⁻¹ in melted ice cores and 3.9 × 10⁴–32.5 × 10⁴ cells ml⁻¹ in brine. Virus to bacterium ratios (VBR) showed a clear seasonal pattern in ice cores with lowest values in winter (range 1.2–20.8), while VBRs in brine were lower (0.2–4.9). Lytic viral production range from undetectable to 2.0 × 10⁴ viruses ml⁻¹ h⁻¹ in ice cores with maximum rates in September and November. In brine maximum, lytic viral production occurred in November (1.18 × 10⁴ viruses ml⁻¹ h⁻¹). Low burst sizes were typical (3.94–4.03 viruses per bacterium in ice cores and 3.16–4.0 viruses per bacterium in brine) with unusually high levels of visibly infected cells—range 40–50%. This long-term investigation revealed that viral activity was apparent within the sea ice throughout its annual cycle. The findings are discussed within the context of limited data available on viruses in sea ice.     

Transmission electron microscope analysis of virus-like particles in the freshwater lakes of Signy Island, Antarctica

Water samples from a range of fresh-water Antarctic lakes on Signy Island (South Orkney Islands: 60°45′S, 45 °38′W) were examined for the presence of virus-like particles (VLPs) during the 1998/1999 field season. It was discovered that VLPs were ubiquitous, morphologically diverse and abundant, with high concentrations ranging from 4.9 × 10⁶ ml⁻¹ to 3.1 × 10⁷ ml⁻¹. Likely hosts include bacteria, cyanobacteria and eukaryotic algae. In addition, an unusually large virus morphotype was observed with a head diameter 370 × 330 nm and a tail 1.3 μm long. Accepted: 15 May 2000     

Use of clove oil to anaesthetize freshwater amphipods

The use of clove oil as a potential anaesthetic for freshwater amphipods was examined at 20 °C. Individuals of Gammarus minus, a common species in southern Illinois, USA, spanning the entire body size range (4.3–14.3 mm), were used to test four anaesthetic concentrations varying from 1.48 × 10⁻⁴ ml ml⁻¹ to 5.9 × 10⁻⁴ ml ml⁻¹. Small-bodied individuals (mean size = 5.4 mm ± 0.27SE) were used to test additional concentrations, up to 14.7 × 10⁻⁴ ml ml⁻¹, a 10-fold span, to identify potential lethal concentrations. At the lowest concentration, time to anaesthesia and recovery was constant at all body sizes. For the three next higher concentrations, time to anaesthesia decreased with increasing concentration while recovery time increased. Activity of amphipods was not affected by the ethanol carrier. In addition, activity did not differ between amphipods that had recovered from anaesthesia and unexposed amphipods. At clove oil concentrations of 8.84 × 10⁻⁴ ml ml⁻¹ and 14.7 × 10⁻⁴ ml ml⁻¹, mortality was 7 and 40%, respectively, indicating, that 5.9 × 10⁻⁴ ml ml⁻¹ was a safe working concentration. No mortality was observed with Gammarus acherondytes, a federally endangered cave amphipod on which the protocol with 80 μl of stock was used in the field. The method enabled us to obtain information on the endangered amphipod which normally would have required the sacrifice of individuals. Thus, research can continue on species for which population numbers are low and for which basic information is needed to formulate meaningful recovery plans.     

Virus impact on heterotrophic bacterioplankton of water reservoirs

The quantitative distribution of viruses and their impact on heterotrophic bacterioplankton were studied in mesotrophic and eutrophic reservoirs of the Volga and Volga-Baltic waterway. The abundance of planktonic virus particles ranged from 9.4 × 10⁶ to 120 × 10⁶ ml⁻¹ and was from 2.5 to 9 times greater than the bacterial numbers. Production of virioplankton varied from 2.1 × 10⁶ to 132 × 10⁶ particles (ml day)⁻¹ and the population turnover time values were between 0.3 and 11.6 days. The maximum values of numbers and production of virio- and bacterioplankton were observed in the eutrophic Ivan’kovo reservoir. Distribution of the viruses in the Volga reservoirs depended to a significant degree on the number and activity of heterotrophic bacterioplankton. The infected bacteria accounted for 5.5–33.5% of the total bacterial abundance. Phages were an important factor of bacterial mortality. During July to September virus-induced bacterial mortality varied between 6.1 and 40.6% (20.2% on average) of daily bacterioplankton production.     

Effects of water velocity on picoplankton uptake by coral reef communities

In previous experiments, rates of picoplankton uptake into coral communities were controlled by sponge and ascidian biomass. Those experimental communities, however, had relatively few sponges and ascidians. In contrast, turbulent transport of particles into the momentum boundary layers can limit particle removal by layered, dense bivalve populations. In this study, the role of water velocity in controlling particulate nutrient-uptake by rubble communities was evaluated, in which the rubble was more completely covered by sponges and ascidians. Picoplankton uptake was proportional to concentration over a range of cell concentrations from 3.0 × 10⁵ to 9.5 × 10⁵ heterotrophic bacteria ml⁻¹, 4.1 × 10⁴ to 1.2 × 10⁵ Synechococcus sp. ml⁻¹ and 6.3 × 10³ to 1.8 × 10⁴ picoeukaryotes ml⁻¹. The first-order uptake rate constants, normalized to sponge and ascidian biomass, were similar to previous experimental communities. Picoplankton uptake increased 1.6-fold over a 7-fold change in water velocity, 0.05–0.35 m s⁻¹. This increase has been interpreted as a result of higher turbulent transport within the rough coral community (canopy), as indicated by a 1.6-fold increase in the bottom friction with increasing water velocity.     

Bacterioplankton of the Gdansk Basin,Baltic Sea

The numbers, biomass, and production of bacterioplankton were determined in the Russian Sector of the Gdansk Basin (Baltic Sea) in 2007–2009. Significant spatial and temporal variations were determined. During the year, bacterial activity increased with increasing water temperature and higher availability of organic substrates. The lowest bacterial production (0.01–31.63 mg C m⁻³ day⁻¹) was observed in late winter and late autumn, while the highest (0.17–341.70 mg C m⁻³ day⁻¹) occurred in spring and summer. Since bacterial numbers and biomass were found to depend on the weather conditions and the terrigenous inflow, significant variations were observed from year to year. The highest and lowest numbers and biomass of bacterioplankton determined in summer were 0.09–1.10 × 10⁶ cells mL⁻¹ and 2–22 mg C m⁻³ for July 2007 and 1.96–11.23 × 10⁶ cells mL⁻¹ and 23–123 mg C m^–3 for July 2009. The values of these parameters were the highest along the coast and decreased towards the open sea.     

Arctic Sea ice biota: design and evaluation of a mesocosm experiment

A mesocosm experiment (enclosure volume 220 l) was designed such that sea ice inhabited by Arctic Sea ice organisms was formed and maintained under natural conditions at 66°N in Rovaniemi, Finland. The experiment was run from natural freezing in December 1994 to melting in April 1995. The ice was inhabited by diatoms, chlorophyceae, heterotrophic flagellates, ciliates, nematodes and turbellarians. Biomass in the ice, expressed as Chlorophyll a concentration, was 20–110 μg l⁻¹; total cell densities varied from 5 × 10⁶ to 35 × 10⁶ cells l⁻¹. Amongst phototrophic organisms, a succession from a flagellate-dominated community (Chlamydomonas sp.) to a multi-species diatom-dominated community was observed. Typical Arctic species such as Nitzschia frigida and Melosira arctica were present in the ice. Bacterial concentration varied between 2 × 10⁸ and 7 × 10⁸ cells l⁻¹. At least two trophic levels were present in the ice. Received: 3 April 1997 / Accepted: 9 September 1997     

Viral dynamics and patterns of lysogeny in saline Antarctic lakes

Antarctic lakes are extreme ecosystems with microbially dominated food webs, in which viruses may be important in controlling community dynamics. A year long investigation of two Antarctic saline lakes (Ace and Pendant Lakes) revealed high concentrations of virus like particles (VLP) (0.20–1.26 × 10⁸ ml⁻¹), high VLP: bacteria ratios (maximum 70.6) and a seasonal pattern of lysogeny differing from that seen at lower latitudes. Highest rates of lysogeny (up to 32% in Pendant Lake and 71% in Ace Lake) occurred in winter and spring, with low or no lysogeny in summer. Rates of virus production (range 0.176–0.823 × 10⁶ viruses ml⁻¹ h⁻¹) were comparable to lower latitude freshwater lakes. In Ace Lake VLP did not correlate with bacterial cell concentration or bacterial production but correlated positively with primary production, while in Pendant Lake VLP abundance correlated positively with both bacterial cell numbers and bacterial production but not with primary production. In terms of virus and bacterial dynamics the two saline Antarctic lakes studied appear distinct from other aquatic ecosystems investigated so far, in having very high viral to bacterial ratios (VBR) and a very high occurrence of lysogeny in winter.     

Biofilm formation on brass coupons exposed to a cooling system of an oil refinery

Brass coupons (70% Cu 30% Zn) were exposed to a cooling freshwater system of an oil refinery, in order to investigate susceptibility of the metal to biofilm formation. The coupons were fixed on bypasses at points which allowed the circulation of makeup, cooling and return water. The number of aerobic, anaerobic and sulfate-reducing bacteria was determined in both the planktonic and the sessile phases. Maximum bacterial concentrations were detected in the cooling water, corresponding to 2.1 ± 0.1 × 10⁶ CFU ml⁻¹ (planktonic phase) and 1.3 ± 0.2 × 10⁵ CFU cm⁻² (sessile phase) for aerobic bacteria and to 3.2 ± 0.3 × 10⁵ cells ml⁻¹ (planktonic phase) and 6.2 ± 0.7 × 10⁵ cells cm⁻² (sessile phase) for anaerobic bacteria. Sulfate-reducing bacteria (SRB) were observed only in the planktonic phase, being found in greater numbers in the return water. Scanning electron microscopy (SEM) analysis indicated that biofilm formation occurred at the three monitored sites and showed a diversity in cell morphology. Nonetheless, no evidence of corrosion was observed on the brass coupons during the experimental period. Received 22 May 1997/ Accepted in revised form 19 September 1997     

Virulence of a South African isolate of the <Emphasis Type="Italic">Cryptophlebia leucotreta</Emphasis> granulovirus to <Emphasis Type="Italic">Thaumatotibia leucotreta</Emphasis> neonate larvae

Surface inoculation dose–response and time–response bioassays and detached fruit bioassays were conducted with a novel South African isolate of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) against Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Noctuidae) neonate larvae. LC₅₀ and LC₉₀ values were estimated to be 4.095 × 10³ and 1.185 × 10⁵ OBs ml⁻¹, respectively. LT₅₀ and LT₉₀ values were estimated to be 4 days 22 h and 7 days 8 h, respectively, categorising the virus as a fast or type 2 granulovirus. There was a conspicuous difference in behaviour between larvae on inoculated diet and untreated diet, resulting in a significant reduction in penetration of diet. Bioassays on detached Navel oranges revealed LC₅₀ and LC₉₀ values of 9.310 × 10⁷ and 1.515 × 10⁹ OBs ml⁻¹, when using data on numbers of larvae per fruit rather than on numbers of infested fruit. Field trials will be conducted.     

Occurrence and food consumption of the common eider,Somateria mollissima,in the Wadden Sea of Schleswig-Holstein

The number of eider in the Wadden Sea of Schleswig-Holstein was counted by aerial surveys during 1986 and 1987. The highest number occurred during migration in October 1987 with 151 000 ducks, the lowest number during the breeding time in May 1987 with 6000 ducks. About 100 000–120 000 eiders moult in July/August in the Wadden Sea of Schleswig-Holstein, but only 30 000–40 000 stay over winter. The average number was 62 000 ducks. Eider have increased in number since the seventies, when the average population size was estimated to be only 23 000. The increase referred mainly to moulting and migrating eider, whereas numbers in winter remained constant. There are substantial changes in the spatial distribution over the year. In most areas sites used during moult, migration and winter can be clearly separated, although so far no obvious differences in the morphology of these areas could be found. The annual food consumption was calculated to be 3.1×10⁶ kg AFDW or 1.3 g AFDW×m⁻²×year⁻¹, which is about 5% of the average biomass of macrozoobenthos. The increase in the number of eider has led to a significant increase in total food consumption of carnivorous birds, which was estimated at 7.1×10⁶ kg AFDW × year⁻¹ in the seventies and now reaches 9.0×10⁶ kg AFDW×year⁻¹, of which the eider takes 34%. The reasons for and consequences of the increase of the eider are discussed in context with the eutrophication of the North Sea and possible competition with shellfishery. Presented at the VI International Wadden Sea Symposium (Biologische Anstalt Helgoland, Wattenmeerstation Sylt, D-2282 List, FRG, 1–4 November 1988)     

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus–insect cell expression system

A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O₂ mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6–10 × 10⁶ cells ml⁻¹) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 10⁶ cells ml⁻¹ were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell⁻¹, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11–38 mg L⁻¹ of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 10⁶ cells ml⁻¹. Received 09 December 1996/ Accepted in revised form 13 April 1997     

Protozoan Bacterivory in the Ice and the Water Column of a Cold Temperate Lagoon

Abstract Bacterial abundance and bacterivorous protist abundance and activity were examined in ice-brine and water column communities of a cold temperate Japanese lagoon (Saroma-Ko Lagoon, Hokkaido, 44°N, 144°E), during the late winter phase of ice community development (February–March 1992). Bacterial abundance averaged 6 and 1 × 10⁵ cells ml⁻¹ in the ice-brine and plankton samples, respectively, and generally decreased during the sampling period. Bacterivorous protists, identified based on direct observation of short-term (<1 h) ingested fluorescently labeled bacteria (FLB) in their food vacuoles, were largely dominated by flagellates, mainly cryothecomonad-type and chrysomonad-like cells and small dinoflagellates of the genus Gymnodinium. Bacterivorous ciliates included mainly the prostomatid Urotricha sp., the scuticociliates Uronema and Cyclidium, the choreotrichs Lohmaniella oviformis and Strobilidium, and the hypotrich Euplotes sp. Protist abundance averaged 4 × 10³ and 8.1 cells ml⁻¹ in the ice-brine and 0.3 × 10³ and 1.2 cells ml⁻¹ in the plankton, for flagellates and ciliates, respectively. In contrast to bacteria, the abundance of protists generally increased throughout the sampling period, indicating predator–prey interactions. Protistan bacterivory, measured from the rate of FLB disappearance over 24 h, averaged 36% (ice) and 24% (plankton) of bacterial standing stock and exhibited the same seasonal pattern as for protist abundance. The calculated specific clearance (range, 2–67 nl protozoa⁻¹ h⁻¹) and ingestion (<1–26 particles protozoa⁻¹ h⁻¹) rates were likely to be minimal estimates and grazing impact may have been higher on occasion. Indications for the dependence of ``bacterivorous protists' on nonbacterial food items were also provided. Although alternative sources of bacterial loss are likely to be of importance, this study provides evidence for the potential of protozoan assemblages as bacterial grazers in both sea ice-brine biota and water column at the southern limit of sea ice in the northern hemisphere. Received: 30 July 1998; Accepted: 18 November 1998     

Dynamics of cyanophage-like particles and algicidal bacteria causing Microcystis aeruginosa mortality

The dynamics of cyanophage-like particles and algicidal bacteria that infect the bloom-forming cyanobacterium Microcystis aeruginosa was followed in a hyper-eutrophic pond from September 1998 to August 1999. The densities of M. aeruginosa ranged between 4.0 × 10⁵ and 1.9 × 10⁷ cells ml⁻¹, whereas those of algicidal bacteria were between 4.0 and 5.1 × 10² plaque-forming units (PFU) ml⁻¹ and those of cyanophage-like particles were between <5.0 × 10² and 7.1 × 10³ PFU ml⁻¹. A significant relationship was found between the densities of algicidal bacteria and M. aeruginosa (r = 0.81, n = 69, P < 0.001), suggesting that the dynamics of the algicidal bacteria may regulate the abundance of M. aeruginosa. Occasional peaks of density of cyanophage-like particles were detected in October, June, and August, when sharp declines in M. aeruginosa cell densities were also observed. The densities of cyanophage-like particles became undetectable when the abundance of M. aeruginosa was low, suggesting the density-dependent infection of M. aeruginosa by cyanophage-like particles. Thus, we suggest that infections of both algicidal bacteria and cyanophage-like particles are important biological agents that decompose blooms of M. aeruginosa in freshwater environments. Received: August 31, 2000 / Accepted: December 6, 2000     

Distribution of viruses and their impact on bacterioplankton in mesotrophic and eutrophic reservoirs

Spatial distribution of planktonic viral particles (virioplankton) and mortality of heterotrophic bacteria caused by viral lysis were studied in the eutrophic Ivan’kovskoe and mesotrophic Uglichskoe reservoirs (the Upper Volga). During the summer peak of phytoplankton, the number of viral particles was higher in the Ivan’kovskoe Reservoir ((55.1 ± 9.5) × 10⁶ ml⁻¹ on average) than in the Uglichskoe Reservoir ((42.9 ± 5.1) × 10⁶ ml⁻¹ on average). The ratio of viral to bacterial abundances ranged from 2.5 to 7.0. The average number of mature phages in infected heterotrophic bacteria varied from 17 to 109 particles/cell. Most of the infected bacterial cells in the Ivan’kovskoe Reservoir were rod-shaped, and, in the Uglichskoe Reservoir, they were mainly vibrio-shaped. In the Ivan’kovskoe Reservoir, from 8.3 to 22.4% of planktonic bacteria were infected by phages, suggesting phage-induced mortality of bacterioplankton equal to 10.5–34.8% (19.1% on average) of the daily bacterial production. In the Uglichskoe Reservoir, from 9.4 to 33.5% of bacteria were phage-infected, suggesting phage-induced bacterial mortality of 13.7–40.2% (23.5% on average) of the daily bacterial production. The obtained results testify to an important role of autochthonous viruses in the regulation of bacterioplankton abundance and production in the reservoirs.     

Seasonal changes of Antarctic marine bacterioplankton and sea ice bacterial assemblages

The distributions of bacterial populations in sea ice and underlying seawater were investigated on the continental shelf of the “Terre Adélie” area. A reference station was sampled weekly from January 1991 to January 1992. In winter, the survey included a minimum of six sampling layers: surface and bottom ice, brine, seawater from the interface, and at 0.5 and 2 m depth. In seawater, the total bacterial abundance ranged from 0.5 × 10⁵ cells ml⁻¹ in July to 6.0 × 10⁵ cells ml⁻¹ after ice break. Values reaching 2.5 × 10⁶ cells ml⁻¹ were recorded in the overlying ice cover. Mean cell volumes were twice as high in brine as in seawater. The saprophytic bacterial abundance ranged from 5.0 × 10⁴ CFU (colony-forming units) ml⁻¹ in some winter interface samples to less than 1.0 × 10³ CFU ml⁻¹ in most of the summer seawater samples. In sea ice a clear decreasing gradient for most of the studied bacterial parameters from the surface layers towards the bottom layer was found. The ice cover had a discernible impact on underlying seawater, but its influence was restricted to a limited interface layer.     

Production and characterisation of a biosurfactant isolated from Pseudomonas aeruginosa UW-1

Pseudomonas aeruginosa UW-1 produced 17–24 g L⁻¹ rhamnolipid in vegetable oil-containing media in shake flask cultures in 13 days. In time course studies of growth and rhamnolipid production in a salts medium containing 6% canola oil, total bacterial count reached 2.6 × 10¹⁰ CFU ml⁻¹ after 48 h and a maximum rhamnolipid yield of 24.3 g L⁻¹ was obtained after 9 days. Rhamnolipid components were purified and separated by chloroform-methanol extraction and TLC chromatography. The major rhamnolipid components were characterised as L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate and L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate by nuclear magnetic resonance and mass spectrometry. The components were separated preparatively by silica gel column chromatography. The recovered monorhamnosyl fraction contained no dirhamnosyl moiety while the recovered dirhamnosyl fraction contained 5% of the monorhamnosyl moiety when analyzed by HPLC. The ratio of mono- to dirhamnosyl components produced by P. aeruginosa UW-1 was determined by HPLC to be 4 : 1 by weight. Purified mono- and dirhamnosyl components had the same CMC value of 40 μg ml⁻¹ and decreased the surface tension of water to 27.7 and 30.4 dynes cm⁻¹, respectively. Received 04 April 1997/ Accepted in revised form 15 July 1997     

Degradation of naphthalene by cells of Pseudomonas sp. strain NGK 1 immobilized in alginate, agar and polyacrylamide

A Pseudomonas sp. strain NGK 1 (NCIM 5120) was immobilized in various matrices, namely, alginate, agar (1.8 × 10¹¹ cfu g⁻¹ beads) and polyacrylamide (1.6 × 10¹¹ cfu g⁻¹ beads). The degradation of naphthalene was studied, by freely suspended cells (4 × 10¹⁰ cfu ml⁻¹) and immobilized cells in batches, with shaken culture and continuous degradation in a packed-bed reactor. Free cells brought about the complete degradation of 25 mmol naphthalene after 3 days of incubation, whereas, a maximum of 30 mmol naphthalene was degraded by the bacteria after 3–4 days of incubation with 50 mmol and 75 mmol naphthalene, and no further degradation was observed even after 15 days of incubation. Alginate-entrapped cells had degraded 25 mmol naphthalene after 3.5 days of incubation, whereas agar- and polyacrylamide-entrapped cells took 2.5 days; 50 mmol naphthalene was completely degraded by the immobilized cells after 6–7 days of incubation. Maximum amounts of 55 mmol, 70 mmol and 67 mmol naphthalene were degraded, from an initial 75 mmol naphthalene, by the alginate-, agar- and polyacrylamide-entrapped cells after 15 days of incubation. When the cell concentrations were doubled, 25 mmol and 50 mmol naphthalene were degraded after 2 and 5.5 days of incubation by the immobilized cells. Complete degradation of 75 mmol naphthalene occurred after 10 days incubation with agar- and polyacrylamide-entrapped␣cells, whereas only 60 mmol naphthalene was degraded by alginate-entrapped cells after 15 days of␣incubation. Further, with 25 mmol naphthalene, alginate-, agar- and polyacrylamide-entrapped cells (1.8 × 10¹¹ cfu g⁻¹ beads) could be reused 18, 12 and 23 times respectively. During continuous degradation in a packed-bed reactor, 80 mmol naphthalene 100 ml⁻¹ h⁻¹ was degraded by alginate- and polyacrylamide-entrapped cells whereas 80 mmol naphthalene 125 ml⁻¹␣h⁻¹ was degraded by agar-entrapped cells. Received: 21 October 1997 / Received revision: 15 January 1998 / Accepted: 18 January 1998     

A Mutant of Metarhizium anisopliae var. acridum with Enhanced Submerged Conidiation

Summary An insertional mutant of Metarhizium anisopliae is described with enhanced submerged conidiation. In a 500 ml submerged culture, this mutant produces a mean of 4.05 × 10⁸ propagules ml⁻¹ from an inoculum of 1 × 10⁶ conidia, where the parental strain accumulates only 3.75 × 10⁴ propagules ml⁻¹.     

Stability validation of seeding cell control parameters in large-scale hybridoma cell culture

Stability and reproducibility of seeding cell performance in large-scale hybridoma cell culture has been reported by controlling only initial cell seeding density. The aim of the current study was to integrate multiple seeding cell control parameters to maintain stable and consistent cell physiological status for HAb18 cell expansion. Three parameters and their ranges were investigated, including initial cell seeding density in the range of 0.075–0.5×10⁶ cells ml⁻¹, “timepost” after cell passage between 8 and 36 h, and duration of subculture up to 6 months after cell revival. Cell performance was tested at the 1 L, 5 L, and 75 L scales. Desirable performance was found within the following parameter ranges: initial cell seeding density of 0.1–0.3×10⁶ cells ml⁻¹, “timepost” after cell passage between 14 and 22 h, and duration of subculture within 3 months of cell revival. Our results showed that cell growth rate and antibody productivity of three batches at 1 L, 5 L, and 75 L scale were found to be stably maintained within a range of 0.036–0.047 h⁻¹ and 0.577–0.747 pg cell⁻¹ h⁻¹, with the positivity rate of antigen-binding activity within 97–99.75%, and the intensity of fluorescence around 200. This study may provide a simple but effective method to maintain seeding cell physiological status stable and consistent by combining seeding cell control parameters.