Poly(carbamoylsulphonate) as a matrix for whole cell immobilization - biological characterization

Summary Poly(carbamoylsulphonate) (PCS), a hydrogel matrix of low toxicity for the immobilization of microorganisms was characterized with respect to physiological properties. The survival rates of immobilized Paracoccus denitrificans in PCS were greater than 99 %, the initial division rate of the bacterium inside the gel was the same as of free suspended cells and decreases with increasing cell density within the first days. The polymer has good resistance to microbial degradation and excellent mechanical stability. First results of long term behaviour and kinetic data of nitrifying bacteria entrapped within PCS gel beads are shown.     

Inhibition of the metabolism of nitrifying bacteria by direct electric current

The nitrifying bacteria in activated sludge and biofilms consisting of the bacteria immobilized on polypropylene packing were subjected to an electric current via two electrodes. In activated sludge, the metabolism of nitrifying bacteria was inhibited when the applied current was over 2.5 A m^–2, whilst in biofilms, inhibition began when the current reached 5 A m^–2. At 15 A m^–2, the nitrification rate of NH₄ ⁺-N in a biofilm with a bacterial density of 1.62 g total solids, dry wt m^–2 decreased to about 80% of its initial value. Ninety-two % of the initial biomass on the packing was retained after 36 h.     

Immobilization of nitrifying bacteria in porous pellets of urethane gel for removal of ammonium nitrogen from waste-water

Summary The effects of immobilizing materials on the activity of nitrifying bacteria and removal of ammonium nitrogen (NH₄-N) from waste-water by immobilized nitrifying bacteria were investigated using six urethane prepolymers. With a urethane prepolymer containing 2.27% free isocyanate, a high activity yield of nitrifying bacteria was obtained. There was a drastic improvement over the conventional method of immobilization by acrylamide in the activity yield. Inorganic synthetic waste-water was treated at a high loading rate of 0.24 kg N·m^–3·day^–1. The NH₄-N concentration of the effluent could be reduced to 2 mg·1^–1 or less and the removal was 90%. The life of the pellets in terms of activity was at least 120 days. Offprint requests to: T. Sumino     

High-efficient and synergetic antibacterial nanocomposite hydrogel with quaternized chitosan/Ag nanoparticles prepared by one-pot UV photochemical synthesis

Hydrogel dressings have significant advantages such as absorption of tissue exudate, maintenance of proper moist environment, and promotion of cell proliferation. However, facile preparation method and high-efficient antibacterial hydrogel dressings are still a great challenge. In this study, a facile approach to prepare antibacterial nanocomposite hydrogel dressing to accelerate healing was explored. The hydrogels consisted of quaternized chitosan and chemically cross-linked polyacrylamide, as well as silver nanoparticles (AgNPs) stabilized by chitosan. The synthesis of the hydrogels including the formation of AgNPs and polymerization of acrylamide was accomplished simultaneously under UV irradiation in 1 hour without adding initiator. The hydrogels showed favorable tensile strength of ∼100 kPa with elongation at break over 1000% and shear modulus of ∼10⁴ Pa as well as suitable swelling ratio, which were appropriate for wound dressing. The combination of quaternized chitosan and AgNPs exhibited high-efficient and synergetic antibacterial performance with low cytotoxicity. In vivo animal experiments showed that the hydrogel can effectively prevent wound infection and promote wound healing. This study provides a facile method to produce antibacterial hydrogel wound dressing materials.     

Turnover of “new” and “old” carbon in soil microbial biomass

The contributions of “new” carbon coming from plants with the C4-type of photosynthesis (maize) and “old” carbon from soil organic matter (SOM) formed under C3 vegetation as carbon sources for microorganisms was determined. Soil samples were taken from the plots of field experiments on Chernozem and Phaeozem. The values of δ¹³C were determined in evolved CO₂, SOM, total microbial biomass (C_mic), and phospholipid fatty acids (PLFA), assuming that the PLFA markers for certain taxonomic groups of microorganisms enriched in C4 carbon indicated a more significant role of these microorganisms in the transformation of root exudates and plant residues. Carbon pools were arranged in the following order by the degree of their enrichment with “new” C: SOM < C_mic < CO₂. Consequently, the “new” carbon proved to be a more preferable substrate for microbial growth than the “old” one. The share of C4 in the markers varied from 18 to 60% (on average 38%) in Phaeozem and from 15 to 40% in Chernozem (on average 28%). The groups of microorganisms in Phaeozem were arranged in the following order by the degree of their enrichment with “new” carbon: protozoa < saprotrophic fungi < actinomycetes < gram-positive bacteria < gramnegative bacteria < mycorrhizal fungi. In Chernozem, the contribution of C4 to the carbon composition of PLFA did not differ significantly for various groups of microorganisms. The C4 content within the PLFA markers of fungi and gram-negative bacteria did not demonstrate any crucial contribution of these groups of organisms to the transformation of “new” C. The long-term C3–C4 transition probably results in formation of a broad range of carbon pools similar in their C4 content but different in resistance to mineralization; therefore, gram-positive bacteria could assimilate C4 from resistant C pools. The low content of “new” carbon in the PLFA markers of fungi may be explained by a considerable portion of dormant forms.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Ammonia removal from freshwater using nitrifying bacteria enriched from a seawater aquaculture pond

Intensively cultured fish stock when fed protein-rich feeds typically excrete high concentrations of total ammoniacal-nitrogen (TAN) into the water column which can have adverse effects on productivity, and upon the environment when aquaculture water is discharged. An immobilized culture of nitrifying bacteria isolated from prawn pond water and known to effectively remove TAN from saline water was tested for its ability to remove TAN from freshwater. The culture was readily adaptable to non-saline conditions and maintained TAN at less than 0.25 mg l^–1, even with a daily addition of 3.2 to 4.2 mg TAN l^–1 per d. The use of the immobilized culture of nitrifying bacteria represents an innovative and economical in situ treatment technology for removal of TAN in both saline and freshwater.     

Immobilization of nitrifying bacteria by polyethylene glycol prepolymer

The effects of immobilizing materials on the activity of nitrifying bacteria were investigated by using 11 kinds of prepolymers of polyethylene glycol. Relative respiratory activity of immobilized nitrifying bacteria with polyethylene glycol metacrylate prepolymer was higher than that of polyethylene glycol acrylate prepolymer, and there was a tendency for relative respiratory activity to be higher with a prepolymer of greater molecular weight. With the polyethylene glycol prepolymer, there was a drastic improvement over the conventional method of immobilization by acrylamide in the relative respiratory activity of the pellet. Inorganic synthetic wastewater was treated under a high loading rate of 1.14 kg-N/m³·d. Influent NH₄-N could be removed to 2 mg/l or less and the nitrogen removal was 90%.     

Enhanced efficacy of nitrifying biomass by modified PVA_SB entrapment technique

In this study, we developed a novel technique for preparing polyvinyl alcohol (PVA) hydrogel as an immobilizing matrix by the addition of sodium bicarbonate. This resulted in an increase in the specific surface area of PVA_sodium bicarbonate (PVA_SB) hydrogel beads to 65.23 m² g^?1 hydrogel beads, which was approximately 85 and 14 % higher than those of normal PVA and PVA_sodium alginate (PVA_SA) hydrogel beads, respectively. The D _e value of PVA_SB hydrogel beads was calculated as 7.49 × 10^?4 cm² s^?1, which was similar to the D _e of PVA_SA hydrogel beads but nearly 38 % higher than that of the normal PVA hydrogel beads. After immobilization with nitrifying biomass, the oxygen uptake rate and the ammonium oxidation rate of nitrifying biomass entrapped in PVA_SB hydrogel beads were determined to be 19.53 mg O₂ g MLVSS^?1 h^?1 and 10.59 mg N g MLVSS^?1 h^?1, which were 49 and 43 % higher than those of normal PVA hydrogel beads, respectively. Scanning electron microscopy observation of the PVA_SB hydrogel beads demonstrated relatively higher specific surface area and revealed loose microstructure that was considered to provide large spaces for microbial growth. This kind of structure was also considered beneficial for reducing mass transfer resistance and increasing pollutant uptake.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate Adriamonas peritocrescens on the competition for limiting amounts of ammonium between the ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h^-1. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM. A. globiformis won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h^-1, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 × 10⁵ and 12 × 10⁵ flagellates ml^-1 were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h^-1, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Continuously Percolated Soil Columns

Although the absence of nitrate formation in grassland soils rich in organic matter has often been reported, low numbers of nitrifying bacteria are still found in these soils. To obtain more insight into these observations, we studied the competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi with soil columns containing calcareous sandy soil. The soil columns were percolated continuously at a dilution rate of 0.007 h^-1, based on liquid volumes, with medium containing 5 mM ammonium and different amounts of glucose ranging from 0 to 12 mM.A. globiformis was the most competitive organism for limiting amounts of ammonium. The numbers of N. europaea and N. winogradskyi cells were lower at higher glucose concentrations, and the potential ammonium-oxidizing activities in the uppermost 3 cm of the soil columns were nonexistent when at least 10 mM glucose was present in the reservoir, although 10⁷ nitrifying cells per g of dry soil were still present. This result demonstrated that there was no correlation between the numbers of nitrifying bacteria and their activities. The numbers and activities of N. winogradskyi cells decreased less than those of N. europaea cells in all layers of the soil columns, probably because of heterotrophic growth of the nitrite-oxidizing bacteria on organic substrates excreted by the heterotrophic bacteria or because of nitrate reduction at reduced oxygen concentrations by the nitrite-oxidizing bacteria. Our conclusion was that the nitrifying bacteria were less competitive than the heterotrophic bacteria for ammonium in soil columns but that they survived as viable inactive cells. Inactive nitrifying bacteria may also be found in the rhizosphere of grassland plants, which is rich in organic carbon. They are possibly reactivated during periods of net mineralization.     

Atmospheric methane removal by methane-oxidizing bacteria immobilized on porous building materials

Biological treatment using methane-oxidizing bacteria (MOB) immobilized on six porous carrier materials have been used to mitigate methane emission. Experiments were performed with different MOB inoculated in building materials at high (~20 % (v/v)) and low (~100 ppmv) methane mixing ratios. Methylocystis parvus in autoclaved aerated concrete (AAC) exhibited the highest methane removal rate at high (28.5?±?3.8 μg CH₄ g^?1 building material h^?1) and low (1.7?±?0.4 μg CH₄ g^?1 building material h^?1) methane mixing ratio. Due to the higher volume of pores with diameter >5 μm compared to other materials tested, AAC was able to adsorb more bacteria which might explain for the higher methane removal observed. The total methane and carbon dioxide-carbon in the headspace was decreased for 65.2?±?10.9 % when M. parvus in Ytong was incubated for 100 h. This study showed that immobilized MOB on building materials could be used to remove methane from the air and also act as carbon sink.     

Nitrogen conservation in a tropical rain forest

Summary A series of plots in an Amazonian Rain Forest were trenched and treated with calcium to determine the effects of perturbation on numbers and activity of nitrifying bacteria. Although treatment resulted in between 2 and 26% of the nitrogen being lost from the humus layer, virtually all of it was in the NH ₄ ⁺ form. Numbers of nitrifying bacteria in the plots were relatively low. The low numbers and low activity of nitrifying bacteria is attributed to the low pH and high concentration of tannins in the root mat. It is hypothesized that the suppression of nitrifying bacteria results in nitrogen conservation in the rain forest.     

Estimation of Endocarpon pusillum Hedwig carbon budget in the Tengger Desert based on its photosynthetic rate

This study investigated the photosynthetic rate of the lichen Endocarpon pusillum at the Chinese Academy of Sciences Shapotou Desert Research Station and estimated its annual contribution to the carbon budget in the ecosystem. The software SigmaPlot 10.0 with “Macro-Area below curves” was used to calculate the carbon fixation capacity of the lichen. The total carbon budget (ΣC) of the lichen was obtained by subtracting the respiratory carbon loss (ΣDR) from the photosynthetic carbon gain (ΣNP). Because water from precipitation plays an important role in photosynthesis in this ecosystem, the annual carbon budget of E. pusillum at the station was estimated based on the three-year average precipitation data from 2009 to 2011. Our results indicate that the lichen fixes 14.6 g C m^?2 annually. The results suggest that artificial inoculation of the crust lichen in the Tengger Desert could not only help reduce the sand and dust storms but also offer a significant carbon sink, fixing a total of 438000 t of carbon over the 30000 km² of the Tengger Desert. The carbon sink could potentially help mitigate the atmospheric greenhouse effect. Our study suggests that the carpet-like lichen E. pusillum is an excellent candidate for “Bio-carpet Engineering” of arid and semi-arid regions.     

Effect of Storm Events on Benthic Nitrifying Activity

Storm events resulted in a marked reduction in the benthic nitrifying activity of a stream receiving geothermal inputs of ammonium. Subsequently, nitrifying activity demonstrated a logarithmic increase until a stable activity was reached 10 to 12 days after the storm event. The rate of increase of poststorm nitrifying activity was used to calculate the nitrifier growth rate in situ (0.0075 to 0.0116 h⁻¹) which was lower than the growth rates observed for laboratory cultures of nitrifying bacteria isolated from the sediments (0.0233 to 0.0990 h⁻¹).     

Microscale Distribution of Populations and Activities of Nitrosospira and Nitrospira spp. along a Macroscale Gradient in a Nitrifying Bioreactor: Quantification by In Situ Hybridization and the Use of Microsensors

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 μm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.     

A comparison of serological techniques for detecting and identifying honeybee viruses

The sensitivity and specificity of conventional Ouchterlony gel-diffusion, immuno-osmoelectrophoresis (IO), immune serum electron microscopy (ISEM), “decoration,” radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) tests for detecting black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), and sacbrood virus (SBV) particles in extracts of diseased honeybees were compared. A “slow” ISEM method detected virus particles in extracts of individuals or groups of individuals diluted to 10^?3 and 10^?4, respectively, whereas the IO method and a “fast” ISEM method using protein A were one-tenth as sensitive, and Ouchterlony gel-diffusion tests were only one-thousandth as sensitive. Using the antibody “decoration” technique, mixtures of serologically unrelated virus particles could be resolved. RIA and ELISA were found to be one thousand times more sensitive than ISEM in detecting the particles of BQCV, CBPV, KBV, and SBV; however, nonspecific reactions occurred when using RIA with very dilute particle suspensions, and this made dilution endpoints difficult to assess, but this did not occur when using the ELISA method. There was little difference in the effectiveness of rabbit or hen antisera in the tests, except when protein A was used as it does not combine with hen antibodies.     

Effect of various parameters on viability and growth of bacteria immobilized in sol–gel-derived silica matrices

Immobilized bacteria are being extensively used for metabolite production, biocatalysts, and biosensor construction. However, long-term viability and metabolic activity of entrapped bacteria is affected by several conditions such as their physiological state, the presence of high-osmolarity environments, porous structure and shrinkage of the matrix. The aim of this work was to evaluate the effect of various parameters on bacteria immobilized in sol–gel-derived silica matrices. With this purpose, we evaluated the stress of immobilization over bacteria cultures obtained from different growing states, the effect of cell density and bacteria capability to proliferate inside matrices. Best results to attain longer preservation times were obtained when we immobilized suspensions with an optimized bacterial number of 1 × 10⁷ cfu/gel in the presence of LB medium using aqueous silica precursors. Furthermore, the impact of osmotic stress with the subsequent intracellular trehalose accumulation and the addition of osmolites were investigated. Shorter preservation times were found for bacteria immobilized in the presence of osmolites while trehalose accumulation in stressed cells did not produce changes on entrapped bacteria viability. Finally, nutrient addition in silica matrices was studied indicating that the presence of a carbon source without the simultaneous addition of nitrogen was detrimental for immobilized E. coli. However, when both carbon and nitrogen sources were present, bacteria were able to survive longer periods of time.     

Interactions of Nitrifying Bacteria and Heterotrophs: Identification of a Micavibrio-Like Putative Predator of Nitrospira spp.

Chemolithoautotrophic nitrifying bacteria release soluble organic compounds, which can be substrates for heterotrophic microorganisms. The identities of these heterotrophs and the specificities of their interactions with nitrifiers are largely unknown. In this study, we incubated nitrifying activated sludge with ¹³C-labeled bicarbonate and used stable isotope probing of 16S rRNA to monitor the flow of carbon from uncultured nitrifiers to heterotrophs. To facilitate the identification of heterotrophs, the abundant 16S rRNA molecules from nitrifiers were depleted by catalytic oligonucleotides containing locked nucleic acids (LNAzymes), which specifically cut the 16S rRNA of defined target organisms. Among the ¹³C-labeled heterotrophs were organisms remotely related to Micavibrio, a microbial predator of Gram-negative bacteria. Fluorescence in situ hybridization revealed a close spatial association of these organisms with microcolonies of nitrite-oxidizing sublineage I Nitrospira in sludge flocs. The high specificity of this interaction was confirmed by confocal microscopy and a novel image analysis method to quantify the localization patterns of biofilm microorganisms in three-dimensional (3-D) space. Other isotope-labeled bacteria, which were affiliated with Thermomonas, colocalized less frequently with nitrifiers and thus were commensals or saprophytes rather than specific symbionts or predators. These results suggest that Nitrospira spp. are subject to bacterial predation, which may influence the abundance and diversity of these nitrite oxidizers and the stability of nitrification in engineered and natural ecosystems. In silico screening of published next-generation sequencing data sets revealed a broad environmental distribution of the uncultured Micavibrio-like lineage.     

Nitrate reduction in marine sediment: Pathways and interactions with iron and sulfur cycling

Abstract

In coastal marine sediments, the interactions between NO₃ ^? reduction and transformations of Fe and S compounds often occur in a strong gradient of electron activity ("redoxcline"). Denitrification activity is observed throughout the NC₃ ^?‐containing surface zone, although the reduction step from N₂O to N₂ can be inhibited by H₂S in the “redoxcline.”; Survival of denitrifiers is generally poor in NO₃ ^?‐free, reduced sediment; such populations are likely to employ Fe³⁺ reduction in their energy metabolism. At depth, the sediments often contain a larger capacity for “nitrate ammonification”; (dissimilatory NO₃ ^? reduction to NH₄ ⁺) than for denitrification. The “nitrate ammonification”; is found commonly among fermenting bacteria, although SO₄ ^2? reducers may also be involved. In situ activities observed in whole sediment cores indicate that “nitrate ammonification”; may account for as much as one‐third of the carbon oxidation in organic‐rich sediments. The control of partitioning between denitrification and “nitrate ammonification”; at low NO₃ ^? concentrations is poorly investigated, but the larger metabolic capacity of fermenting and S O₄ ^2?‐reducing baceria in relatively reduced sediment could be important. In addition to bacterial reduction, chemical NO₃ ^? reduction is possible where significant amounts of Fe²⁺ (or H₂S) accumulate in the “redoxcline.”;