Effect of Preliminary Filtration on the Functional Characteristics of Bacterioplankton from Lake Khanka

The dependence of the functional characteristics of bacterioplankton from the loess of Lake Khanka on the pore size of filtering materials was investigated. Soluble organic matter (SOM), bacteria, and bacterial consumers adsorbed on particles suspended in the lake water were found to filter differently depending on the pore size of the filtering material. Filters with pore size 4.5 m (filters II) retained up to 20% of SOM and 20–30% of bacterial cells. Filters III with pore size 2.87 m retained almost 50% of SOM and about 40% of bacteria. The double layer of gauze no. 72 (referred to as filter I) with pores size 40 m was unable to completely retain bacterial consumers. In the case of filtrates I and II, the generation time of bacterioplankton decreased with its increasing average daily concentration. In the case of filtrate III, the generation time of bacterioplankton was minimum and did not depend on its concentration. Oxygen consumption rates per one bacterial cell and per unit biomass in filtrates increased with decreasing pore size of the filters through which they had passed. The bacterial biomass and oxygen consumption rate increased exponentially in filtrates III and logarithmically in filtrates I.     

Effect of Protistan Grazing on the Frequency of Dividing Cells in Bacterioplankton Assemblages

Grazing by phagotrophic flagellates and ciliates is a major source of mortality for bacterioplankton in both marine and freshwater systems. Recent studies have demonstrated a positive relationship between clearance rate and prey size for bacterivorous protists. We tested the idea that, by selectively grazing the larger (more actively growing or dividing) cells in a bacterial assemblage, protists control bacterial standing stock abundances by directly cropping bacterial production. Samples of estuarine water were passed through 0.8-μm-pore-size filters (bacteria only) or 20-μm-mesh screens (bacteria and bacterivorous protists) and placed in dialysis tubing suspended in 7 liters of unfiltered water. Changes in total bacterial biovolume per milliliter (bacterial biomass), frequency of dividing cells (FDC), and average per cell biovolume were followed over a period of 24 h. In three experiments, the FDC increased more rapidly and attained higher values in water passed through 0.8-μm-pore-size filters (average, 5.1 to 8.9%; maximum, 15.5%) compared with FDC values in water passed through 20-μm-mesh screens (average, 2.7 to 5.3%; maximum, 6.7%). Increases in bacterial biomass per milliliter lagged behind increases in FDC by about 4 to 6 h. Grazed bacterial assemblages were characterized by lower total biomasses and smaller average cell sizes compared with those of cells in nongrazed assemblages. We conclude that bacterivorous protists control bacterial standing stock abundances partly by preferentially removing dividing cells. Selective grazing of the more actively growing cells may also explain, in part, the ability of slow-growing cells to persist in bacterioplankton assemblages.     

Comparison of different filter types on chlorophyll-a retention and nutrient measurements

In studies on the biochemical compounds in phytoplankton, water samples generally are (pre-) filtered to retain the organisms for extraction. Such filters can be used for further investigations in microscopic or chromatographic (for example High-Performance-Liquid-Chromatography, HPLC) methods, while the filtrates can be used for nutrient or fluorometric measurements as well as for microscopic examinations. Which filter is chosen for a study often depends on its pore size, the costs and, in particular for HPLC measurements, on its chemical compatibility. In our study we compared the chlorophyll-a retention on the filters by HPLC as well as the fluorescence before and after filtration, and nutrient content of the filtrates. The filters we tested were of different material and with various pore sizes. Although Whatman GF/C and GF/F filters are preferred in phytoplankton studies, we found that the Nylon Membrane filter of 0.2 μm pore size provided the most consistent results in chlorophyll-a retention and the one of 0.45 μm pore size in nutrient investigations.     

Purification of Chlamydia trachomatis by a simple and rapid filtration method

A simple method for filter purification of Chlamydia trachomatis from cell culture is described. Crude homogenates of chlamydiae-infected cells were passed through a glass prefilter and a 0.6 microns pore diameter polycarbonate filter. The filtrate was then passed through a 0.2 microns pore diameter filter on which the chlamydiae were trapped. This filter was then back-washed to collect the organisms. These procedures removed cell debris and soluble protein, and yielded particles with a narrow size distribution. The mean yield of viable chlamydiae purified by filtration was 64% when the filters were washed at each stage of the process.     

Seasonal changes in percentages of attached bacteria enumerated in a tidal and a stagnant coastal basin: relation to bacterioplankton productivity

Abstract As part of an extensive ecosystem study, the fate of the bacterioplankton community was followed in the tidal Oosterschelde basin and in the stagnant but saline Lake Grevelingen, The Netherlands. At regular intervals, bacterioplankton biomass, percentages of bacteria attached to particles, bacterioplankton productivity and oxygen consumption rates, together with a number of environmental parameters, were determined. With respect to biomass, productivity and oxygen consumption rates, no significant differences were observed between the two coastal basins. However, the mean percentages of attached bacteria were high in the tidal Oosterschelde basin compared to the stagnant Lake Grevelingen. This could be explained by the observed difference in amounts of colonizable particles. On an annual basis, however, the percentages of attached bacteria were not significantly correlated with the amounts of suspended particles at the sampling stations. A significant, but negative correlation was found between the percentages of attached bacteria and the bacterioplankton productivities at the sampling stations. A probable mechanism for such a negative correlation is discussed.
By size fractionation experiments, bacterioplankton contribution to overall oxygen consumption rates was determined. These contributions amounted to 58% and 94% in spring and winter, respectively. From specific bacterioplankton dissimilation and assimilation rates, mean bacterioplankton carbon conversion coefficients of 26% and 6% were calculated in spring and winter, respectively. These percentages are discussed in the light of probable seasonal differences in the supply of substrates for the bacterioplankton community.     

Use of Membrane Filter Technique in the Microbiological Control for the Brewing Industry

A physical method was developed involving serial filtration with membrane filters for separating yeast cells from bacteria. Such a method eliminates the need for antibiotics previously required to permit differential counting of such populations. All yeast cells filtered were successfully retained and cultivated on a 1.2-mu membrane filter by use of a synthetic medium. All bacteria filtered avoided entrapment on a 1.2-mu membrane filter and were successfully retained and cultivated on a 0.22-mu membrane filter with the same synthetic medium. Final filtrates from these serial filtrations were free from all yeast cells and bacteria when tested with Fluid Thioglycollate Medium.     

Clarification of yeast cell suspensions by depth filtration

Depth filtration can be very attractive for initial clarification because of low capital costs and ease of operation. However, there is currently no fundamental understanding of the effects of the filter pore size and morphology on the overall capacity and filtrate quality. The objective of this study was to examine the flux, capacity, and filtrate turbidity of a series of depth filters with different pore size ratings and multilayer structures for the filtration of yeast cell suspensions. Data were analyzed using available fouling models to obtain insights into the flux decline mechanisms. Filters with small pore size provide high filtrate quality at low capacity, with the reverse being true for the larger pore sizes. The multilayer structure of commercial depth filters leads to improved performance, although the choice of layer properties is critical. The highest capacity was achieved using a multilayer filter in which the upper layer allows significant yeast cell penetration into the filter matrix but still protects the retentive layer that is needed for a high quality filtrate.     

Filtration removal of endotoxin (pyrogens) in solution in different states of aggregation.

Bacterial lipopolysaccharides are recognized as the major cause of pyrogenic reactions from parenteral solutions. Molecular filtration was used to remove these pyrogenic molecules (endotoxins) from contaminated parenteral solutions. Because bacterial lipopolysaccharides can exist in different states of aggregation, depending on the composition of the solution they are suspended in, the full range of possible states of aggregation was examined by using filters with a wide range of pore sizes. Filters of different pore sizes retained endotoxin lipopolysaccharide presumed to be in the vesicle form, the micelle form, or the detergent-solubilized form in aqueous solutions. Endotoxins (pyrogens) were successfully removed from artificially contaminated solutions of concentrated antibiotics by using filters of 10,000-nominal-molecular-weight limit.     

Filtration removal of endotoxin (pyrogens) in solution in different states of aggregation.

Bacterial lipopolysaccharides are recognized as the major cause of pyrogenic reactions from parenteral solutions. Molecular filtration was used to remove these pyrogenic molecules (endotoxins) from contaminated parenteral solutions. Because bacterial lipopolysaccharides can exist in different states of aggregation, depending on the composition of the solution they are suspended in, the full range of possible states of aggregation was examined by using filters with a wide range of pore sizes. Filters of different pore sizes retained endotoxin lipopolysaccharide presumed to be in the vesicle form, the micelle form, or the detergent-solubilized form in aqueous solutions. Endotoxins (pyrogens) were successfully removed from artificially contaminated solutions of concentrated antibiotics by using filters of 10,000-nominal-molecular-weight limit.     

Thymidine Incorporation by Free-Living and Particle-Bound Bacteria in a Eutrophic Dimictic Lake

The percentage of [methyl-³H]thymidine incorporated into samples from a dimictic eutrophic lake and retained on polycarbonate membranes of 3.0-, 1.0-, and 0.2-μm pore size was studied in a lake with filamentous cyanobacteria as the dominant phytoplankton type throughout the period of thermal stratification. Water samples were also examined by epifluorescence microscopy for evidence of algal senescence and bacterial colonization of intact and damaged cyanobacterial filaments. A small percentage (2 to 20%) of bacterial activity was retained by filters with pore sizes ≥ 1 μm in epilimnetic samples. Epilimnetic samples also had a small percentage of cyanobacterial filaments, either intact or damaged, which were visibly colonized by bacteria in summer and fall samples. A significant proportion (20 to 35%) of bacterial activity was retained by filters with pore sizes ≥ 1 μm in samples collected from the metalimnion and hypolimnion during late summer and fall. The proportion of damaged cyanobacterial filaments was higher in these samples than in those from the epilimnion or from those obtained early in the summer. Furthermore, the filaments in these samples were more heavily colonized by bacteria. Overall, particle-bound production accounted for only 2 to 19% of total bacterial production from April to August in all water layers. It appears that the supply of colonizable particles (damaged cyanobacterial filaments) is an important factor affecting the level of particle-bound bacterial activity in this lake.     

Malachite green-INT (MINT) method for determining active bacteria in sewage.

A membrane filtration method was developed to determine the proportion of active (respiring) bacteria at various stages of sewage treatment. Samples were incubated in the presence of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) and, after fixation, passed through membrane filters. Filters were counterstained with malachite green and then were examined by bright-field microscopy. The contrast between bacteria and the filter background was greatly improved by drying and then clearing the filter before counterstaining. By this method, it was found that active bacterial fractions in raw sewage, settled sewage, and secondary effluent were 40, 29, and 58%, respectively, whereas the proportion of respiring bacteria in chlorinated secondary effluent was 6.1%. The active bacterial fraction of activated sludge was found to be 16%. The proposed method represents a significant improvement in speed and simplicity over existing methods for determining active bacteria in sewage.     

Ciliates are the Dominant Grazers on Pico- and Nanoplankton in a Shallow,Naturally Highly Eutrophic Lake

Abundance and biomass of the microbial loop members [bacteria, heterotrophic nanoflagellates (HNF), and ciliates] were seasonally measured in the naturally eutrophic and shallow (2.8 mean depth) Lake Võrtsjärv, which has a large open surface area (average 270 km²) and highly turbid water (Secchi depth <1 m). Grazing rates (filter feeding rates) on 0.5-, 3-, and 6-μm-diameter particles were measured to estimate pico- and nanoplankton grazing (filter feeding) by micro- and metazooplankton. Among grazers, HNF had a low abundance (<50 cells mL^?1) and, due to their low specific filtering rates, they only grazed a minor fraction of the bacterioplankton (≤4.2% of total grazing). Ciliates were relatively abundant (≤158 cells mL^?1) and, considering their high specific feeding rates, were able to graze more than 100% of the bacterial biomass production in the open part of the lake, whereas the average daily grazing accounted for 9.3% of the bacterial standing stock. Ciliates were potentially important grazers of nanoplanktonic organisms (on average, approximately 20% of the standing stock of 3-μm-size particles was grazed daily). Metazooplankton grazed a minor part of the bacterioplankton, accounting for only 0.1% of standing stock of bacteria. Grazing on nanoplankton (3–6 μm) by metazooplankton was higher (0.4% of standing stock). The hypothesis is proposed that ciliates dominate due to a lack of top–down regulation by predators, and HNF have a low abundance due to strong grazing pressure by ciliates.     

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

For indirect DNA extraction for metagenomics studies, bacterial cells can be effectively separated from sample debris by using a simple size exclusion technique, such as filtration, and thereafter lysed. The requirement for the optimal recovery of cells in filtrates is critical to achieve sufficient DNA yield and a representative population. Particles smaller than the filter pore size are expected to be found in the filtrate, whereas particles larger than the filter pore sizes are excluded. However, this is not always the case. It is established that the membrane pore size influences filtration efficiency to some degree. In addition the physicochemical characteristics of the filter suspension and characteristics of the microbial cells being filtered influence the exclusion property of a membrane. This review provides an overview of membrane filtration techniques and the factors that affect filterability of bacteria cells through a filter membrane. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:853–866, 2015     

The use of 14C-labeled bacteria as a tracer of ingestion and metabolism of bacterial biomass by microbial grazers

A method for radiolabeling marine bacteria with d-[U-¹⁴C] glucose and a radiotracer method for measuring ingestion and metabolism of bacterial biomass by ciliated protozoa and other microzooplankton are presented. Problems associated with using live bacterial tracers, i.e., label retention, label recycling, tracer cell size and morphology, and intracellular distribution of label are evaluated.Bacterioplankton assemblages collected from field samples incorporated and retained label as efficiently as coastal isolates which were selected for glucose incorporation. Under grazing experimental conditions, labeled bacteria retained a high proportion of the label (hourly net loss = 1.2%). Bacterial recycling of released dissolved organic ¹⁴C (DO¹⁴C) was 0–2.2% of total ¹⁴C per h. Addition of labeled assemblages to nearshore water samples did not detectably alter mean cell size or size frequency distribution of the attendant bacterioplankton assemblages.In grazing experiments with cultured ciliates (Euplotes sp. and Uronema sp.), radioassay parameters varied as direct functions of predator and prey concentrations. Microautoradiographic analysis corroborated that ¹⁴C incorporation measured in the radioassay by filtration techniques primarily represented ingested bacterial biomass and that problems associated with attached and adsorbed labeled bacteria were minimized. Grazing experiments conducted with bacteria labeled with [U-¹⁴C]glucose yielded ingestion rates comparable to bacteria labeled with [U-¹⁴C]thymidine and additionally provided respiration and exudation rates.     

Interference of tooth differentiation with interposed filters

The effect of interposed Nuclepore filters on the epithelio-mesenchymal interaction in embryonic mouse tooth was studied. Filters with pore sizes of 0.6 and 0.2 μm allowed differentiation of odontoblasts and ameloblasts in the bell-stage tooth germ. This differentiation progressed more rapidly when the 0.6-μm pore size filter was used. Nuclepore filters with 0.1-μm pores prevented differentiation. Electron microscopic examination revealed penetration of cell processes into the filter pores. Cytoplasmic material could be seen in the 0.6-μm pore-size filter within 3 days of cultivation, whereas, in the 0.2-μm filter pores, penetration was slight. After 6 days of cultivation, cytoplasmic material was found at all levels of the 0.2-μm pore-size filter, but not in the channels of the 0.1-μm pore-size filters, preventing differentiation. It is concluded that the 0.1-μm pore-size filter blocks tooth development at the level of mesenchymal cell differentiation into odontoblasts. It is suggested that this differentiation requires a close association between the interacting mesenchymal and epithelial cells.     

Linking the composition of bacterioplankton to rapid turnover of dissolved dimethylsulphoniopropionate in an algal bloom in the North Sea

The algal osmolyte, dimethylsulphoniopropionate (DMSP), is abundant in the surface oceans and is the major precursor of dimethyl sulphide (DMS), a gas involved in global climate regulation. Here, we report results from an in situ Lagrangian study that suggests a link between the microbially driven fluxes of dissolved DMSP (DMSPd) and specific members of the bacterioplankton community in a North Sea coccolithophore bloom. The bacterial population in the bloom was dominated by a single species related to the genus Roseobacter , which accounted for 24% of the bacterioplankton numbers and up to 50% of the biomass. The abundance of the Roseobacter cells showed significant paired correlation with DMSPd consumption and bacterioplankton production, whereas abundances of other bacteria did not. Consumed DMSPd (28 nM day⁻¹) contributed 95% of the sulphur and up to 15% of the carbon demand of the total bacterial populations, suggesting the importance of DMSP as a substrate for the Roseobacter -dominated bacterioplankton. In dominating DMSPd flux, the Roseobacter species may exert a major control on DMS production. DMSPd turnover rate was 10 times that of DMS (2.7 nM day⁻¹), indicating that DMSPd was probably the major source of DMS, but that most of the DMSPd was metabolized without DMS production. Our study suggests that single species of bacterioplankton may at times be important in metabolizing DMSP and regulating the generation of DMS in the sea.     

Certification of Probability of Sterilization of Liquid by Filtration

Four types of hydrosol filters, two reusable (diatomaceous cylinder and fritted-glass funnel) and two disposable (asbestos pad and membrane filter) were challenged with a heavy bacterial suspension to assess their ability to produce sterile filtrates. Two of the four diatomaceous earth filters, the four fritted-glass funnels, and all of the asbestos pads tested generally gave sterile filtrates. However, only one type of filter, one of the membranes in its manufacturer's own holder, consistently gave sterile filtrates. The two other types of membranes usually gave sterile filtrates if tested in one manufacturer's holder, but all types invariably gave contaminated filtrates when tested in another manufacturer's holder. Contaminated filtrates were generally attributed to a poor reusable filter or to a faulty holder used with a disposable filter. If a high degree of certainty is required for sterile heat-labile filtrate, it is suggested that the liquid be passed through two or more filters in a previously tested and proven system.     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

Membrane Filtration of the Reiter Treponeme

A membrane filtration technique with commercially available membrane filters (Millipore Corp.) was effective for the removal of Reiter treponemes from liquids such as fluorescent-antibody conjugates, to which the organisms are added for adsorption. Reiter treponemes from an 8-day culture were not microscopically detectable in filtrates through membranes with a pore diameter of 0.45 μm, but treponemes were demonstrated in the filtrate by cultural methods. No organisms of the 8-day culture passed through a membrane filter having a pore size of 0.22 μm, as determined by microscopy and culture. Culture data indicated that a filter with a pore size of 0.1 μm was necessary to prevent passage of treponemes from 4-day cultures. It is recommended that a membrane filter with a pore size of 0.22 μm or smaller be used for the removal of Reiter treponemes from suspensions and that the age of the culture be considered in choosing filter pore size.