Characteristics of extracellular polymeric substances from sludge and biofilm in a simultaneous nitrification and denitrification system under high salinity stress

The composition and distribution of extracellular polymeric substance (EPS) both from suspended sludge and attached biofilm were investigated in a simultaneous nitrification and denitrification (SND) system with the increase of the salinity from 1.0 to 3.0 %. Fourier-transform infrared (FTIR) spectroscopy and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy were used to examine proteins (PN), polysaccharides (PS) and humic substances (HS) present in EPS. High total nitrogen removal (above 83.9 %) via SND was obtained in the salinity range of 1.0–2.5 %. Total EPS in the sludge increased from 150.2 to 200.6 mg/gVSS with the increase of salinity from 1.0 to 3.0 %, whereas the corresponding values in the biofilm achieved the maximum of 288.6 mg/g VSS at 2.0 % salinity. Dominant composition of EPS was detected as HS in both sludge and biofilm, having the percentages of 50.6–68.6 and 41.1–69.9 % in total EPS, respectively. Both PN and PS contents in soluble EPS (S-EPS), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) of sludge and biofilm increased with the increased salinity. The FTIR spectrum and 3D-EEM fluorescence spectroscopy of S-EPS, LB-EPS and TB-EPS in the sludge and biofilm showed the changes of functional groups and conformations of the compositions in EPS with the increase of salinity. The results demonstrated that the characteristics of EPS varied from sludge to biofilm. The obtained results could provide a better understanding of the salinity effect on the EPS characteristics in a SND system.     

In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging

In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio in Escherichia coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.     

Microbial transformation of biomacromolecules in a membrane bioreactor: implications for membrane fouling investigation

Background

The complex characteristics and unclear biological fate of biomacromolecules (BMM), including colloidal and soluble microbial products (SMP), extracellular polymeric substances (EPS) and membrane surface foulants (MSF), are crucial factors that limit our understanding of membrane fouling in membrane bioreactors (MBRs).

Findings

In this study, the microbial transformation of BMM was investigated in a lab-scale MBR by well-controlled bioassay tests. The results of experimental measurements and mathematical modeling show that SMP, EPS, and MSF had different biodegradation behaviors and kinetic models. Based on the multi-exponential G models, SMP were mainly composed of slowly biodegradable polysaccharides (PS), proteins (PN), and non-biodegradable humic substances (HS). In contrast, EPS contained a large number of readily biodegradable PN, slowly biodegradable PS and HS. MSF were dominated by slowly biodegradable PS, which had a degradation rate constant similar to that of SMP-PS, while degradation behaviors of MSF-PN and MSF-HS were much more similar to those of EPS-PN and EPS-HS, respectively. In addition, the large-molecular weight (MW) compounds (>100 kDa) in BMM were found to have a faster microbial transformation rate compared to the small-MW compounds (<5 kDa). The parallel factor (PARAFAC) modeling of three-dimensional fluorescence excitation-emission matrix (EEM) spectra showed that the tryptophan-like PN were one of the major fractions in the BMM and they were more readily biodegradable than the HS. Besides microbial mineralization, humification and hydrolysis could be viewed as two important biotransformation mechanisms of large-MW compounds during the biodegradation process.

Significance

The results of this work can aid in tracking the origin of membrane foulants from the perspective of the biotransformation behaviors of SMP, EPS, and MSF.     

温带草甸草原土壤N2O产生过程研究

以大量的室内模拟培养实验,以内蒙古温带草甸草原土壤为研究对象,利用AIM乙炔抑制法,模拟野外条件对原样土壤样品进行N₂O产生过程进行研究。实验结果表明:内蒙古温带草甸草原土壤N₂O产生过程以硝化作用为主。其中异养硝化作用起主导作用,自养硝化潜势和反硝化潜势在草原植物不同生长季节变化不同,总体上异养硝化潜势>自养硝化潜势>反硝化潜势。由于自养硝化作用在不同季节的发生,使得草甸草原土壤N₂O的产生潜势也高、低起伏变化。从而揭示了内蒙古温带草原土壤以异养硝化作用过程为主产生N₂O和N₂O排放通量较低的微生物学机理。     

Analysis of Microbial Population Dynamics in a Partial Nitrifying SBR at Ambient Temperature

In this study, a lab-scale partial nitrifying sequencing batch reactor (SBR) was developed to investigate partial nitrification at ambient temperature (16–22 °C). Techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and fluorescence in situ hybridization (FISH) were utilized simultaneously to study microbial population dynamics. Partial nitrification was effectively achieved in response to shifts of influent ammonium concentrations. DGGE results showed that higher ammonia concentration referred to lower ammonia-oxidizing bacteria (AOB) diversity in the SBR. Phylogenetic analysis revealed that all the predominant AOB was affiliated with Nitrosomonas genus. FISH analysis illustrated AOB was the predominant nitrifying bacteria of microbial compositions when SBR achieved partial nitrification (PN) at ambient temperature.     

The relative importance of autotrophic and heterotrophic nitrification in a conifer forest soil as measured by 15N tracer and pool dilution techniques

The importance of heterotrophic nitrification was studied in soil from a mixed-conifer forest. Three sites in the forest were sampled: a clear cut area, a young stand and a mature stand. In the mature stand, the mineral soil (0–10 cm) and the organic layer were sampled separately. Gross rates of N mineralization and nitrification were measured by¹⁵NH ₄ ⁺ and¹⁵NO ₃ ^– isotopic pool dilution, respectively. The rates of autotrophic and heterotrophic nitrification were distinguished by use of acetylene as a specific inhibitor of autotrophic nitrification. In samples supplemented with¹⁵NH ₄ ⁺ and treated with acetylene, no¹⁵NO ₃ ^– was detectable showing that the acetylene treatment effectively blocked the autotrophic nitrification, and that NH ₄ ⁺ was not a substrate for heterotrophic nitrification. In the clear cut area, autotrophic nitrification was the most important NO ₃ ^– generating process with total nitrification (45 ug N kg^–1h^–1) accounting for about one-third of gross N mineralization (140 ug N kg^–1 h^–1). In the young and mature forested sites, gross nitrification rates were largely unaffected by acetylene treatment indicating that heterotrophic nitrification dominated the NO ₃ ^– generating process in these areas. In the mature forest mineral and organic soil, nitrification (heterotrophic) was equal to only about 5% of gross mineralization (gross mineralization rates of 90 ug N kg^–1 h^–1 mineral; 550 ug N kg^–1 h^–1 organic). The gross nitrification rate decreased from the clear cut area to the young forest area to the mineral soil of the mature forest (45; 17; 4.5 ug kg^–1 h^–1 respectively). The¹⁵N isotope pool dilution method, combined with acetylene as an inhibitor of autotrophic nitrification provided an effective technique for assessing the importance of heterotrophic nitrification in the N-cycle of this mixed-conifer ecosystem.     

Extracellular polysaccharides from Desulfovibrio desulfuricans and Pseudomonas fluorescens in the presence of mild and stainless steel

Summary This communication reports the presence of polysaccharides in biofilms formed by pure and mixed cultures of Desulfovibrio desulfuricans and Pseudomonas fluorescens on mild and stainless steel surfaces. The results of colorimetric assays, indicating significant differences between the amounts of neutral sugars present in these biofilms, were supported by gas chromatographic (GC)-mass spectrophotometric and GC-flame ionisation detection analyses. Neutral sugars in biofilms grown on mild steel surfaces were identified and quantified, revealing glucose as a major carbohydrate followed by mannose and galactose in all types of biofilm. Extracellular polymeric substances (EPS) precipitated from bacterial cultures grown with and without steel surfaces were also analysed for their carbohydrate content. The influence of the surfaces present in the cultures on the amount and type of sugars released into the bulk phase was established. There was significantly more carbohydrate in EPS harvested from pure and mixed cultures of D. desulfuricans incubated mild and stainless steel coupons than in EPS obtained from coupon-free cultures. No significant difference in sugar quantities was observed in EPS precipitated from cultures of P. fluorescens grown under different conditions (absence or presence of steel surfaces). The main carbohydrates identified in all types of EPS samples were mannose, glucose and galactose in order of prevalence. Offprint requests to: I. B. Beech     

Functional responses of stream biofilms to flow cessation,desiccation and rewetting

1. Mediterranean climate regions are characterised by long summer droughts that usually involve flow intermittency in low‐ to mid‐order streams. Flow intermittency implies flow cessation, drying and subsequent rewetting of the streambed, and affects both autotrophic and heterotrophic processes. The balance between these processes, as well as the balance in the use of carbon (C), nitrogen (N) and phosphorus (P) may change because of the ongoing increase in stream flow intermittency caused by global change in many regions. It is therefore crucial to understand better the consequences of this phenomenon. 2. Our two initial hypotheses were (i) that flow intermittency would impact more on autotrophic than on heterotrophic processes in stream biofilms owing to the higher water dependence of autotrophs, as well as differences in the water storage capacity of the stream biofilm compartments where autotrophic and heterotrophic processes mainly occur (surface cobbles versus hyporheic sediments) and (ii) that the C‐N‐P use by biofilms would change during the dry period (terrestrial phase) owing to the extreme water stress conditions. These hypotheses were tested by analysing the functional response of the main stream biofilms (epilithic, epipsammic and hyporheic) during flow cessation, desiccation and rewetting in a Mediterranean forested stream. The autotrophic response was characterised through changes in the photon yield, whereas the heterotrophic response was characterised by changes in the extracellular enzyme activities. 3. Streambed desiccation had clear effects on the functioning of stream biofilms. Autotrophic biomass decreased by 80% with streambed desiccation, but recovered rapidly after flow resumption. Heterotrophs were more resistant to water stress, especially in the epipsammic and hyporheic biofilms where bacterial cell density decreased only by 20%. 4. Extracellular enzyme activities remained relatively high, and the balance in the C‐N‐P use by biofilms changed during the dry period. The C and P breakdown capacities were maintained during dry conditions, especially in the epipsammic and hyporheic biofilms, but the degradation of N compounds sharply decreased. Elemental molar ratios (C:N and C:P) of the different biofilms also changed with streambed desiccation. C:P ratios increased from 80 to 300, while the C:N ratios increased from 10 to 16. 5. Given the contrasting responses of autotrophic and heterotrophic processes in the different biofilms, our results suggest that the current increase in flow intermittency extent is likely to increase the relative importance of heterotrophic processes in stream ecosystems, as well as the relative contribution of the hyporheic biofilm to C‐N‐P use. Our results further suggest that the longer streams remain dry, the more the biofilm stoichiometry will change.     

Characterization of extracellular polymeric substances from biofilm in the process of starting-up a partial nitrification process under salt stress

In this study, the characteristics of extracellular polymeric substance (EPS) fractions of biofilm during the process of establishing a partial nitrification under salt stress were analyzed in terms of concentrations, molecular weight distribution, and three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. A partial nitrification was formed successfully with a salinity of 1%. Results indicated that the amount of total EPS increased from 54.2 mg g⁻¹ VSS⁻¹ on day 1 to 99.6 mg g⁻¹ VSS⁻¹ on day 55 due to the NaCl concentration changed from 0 to 10.0 g L⁻¹ in a biofilm reactor. The changes of loosely bound EPS (LB-EPS) compounds under different salt concentrations appeared to be more significant than those of the tightly bound EPS. A clear release of polysaccharides in the LB-EPS fraction was detected during the enhancement of salinity. This was considered as a protective response of bacteria to the salinity. Three fluorescence peaks were identified in the EEM fluorescence spectra of the EPS fraction samples. Two peaks were assigned to the protein-like fluorophores, and the third peak was located at the excitation/emission wavelengths of 275 nm/425–435 nm of the spectra of EPS fractions till the salinity maintained constant at 1%. This information is valuable for understanding the characteristics of EPS isolated from biomass in a saline nitrogen removal system.     

Effects of Xylitol on Xylitol-Sensitive Versus Xylitol-Resistant Streptococcus mutans Strains in a Three-Species in Vitro Biofilm

We studied the effects of xylitol on biofilms containing xylitol-resistant (Xr) and xylitol-sensitive (Xs) Streptococcus mutans, Actinomyces naeslundii and S. sanguinis. The biofilms were grown for 8 and 24 h on hydroxyapatite discs. The viable microorganisms were determined by plate culturing techniques and fluorescence in situ hybridization (FISH) was performed using a S. mutans-specific probe. Extracellular cell-bound polysaccharides (EPS) were determined by spectrofluorometry from single-species S. mutans biofilms. In the presence of 5 % xylitol, the counts of the Xs S. mutans decreased tenfold in the young (8 h) biofilm (p < 0.05) but no effect was seen in the mature (24 h) biofilm. No decrease was observed for the Xr strains, and FISH confirmed these results. No differences were detected in the EPS production of the Xs S. mutans grown with or without xylitol, nor between Xr and Xs S. mutans strains. Thus, it seems that xylitol did not affect the EPS synthesis of the S. mutans strains. Since the Xr S. mutans strains, not inhibited by xylitol, showed no xylitol-induced decrease in the biofilms, we conclude that growth inhibition could be responsible for the decrease of the counts of the Xs S. mutans strains in the clinically relevant young biofilms.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

The contribution of nitrogen transformation processes to total N2O emissions from soils used for intensive vegetable cultivation

The rapid expansion of intensively farmed vegetable fields has substantially contributed to the total N₂O emissions from croplands in China. However, to date, the mechanisms underlying this phenomenon have not been completely understood. To quantify the contributions of autotrophic nitrification, heterotrophic nitrification, and denitrification to N₂O production from the intensive vegetable fields and to identify the affecting factors, a ¹⁵N tracing experiment was conducted using five soil samples collected from adjacent fields used for rice-wheat rotation system (WF), or for consecutive vegetable cultivation (VF) for 0.5 (VF1), 6 (VF2), 8 (VF3), and 10 (VF4) years. Soil was incubated under 50% water holding capacity (WHC) at 25°C for 96 h after being labeled with ¹⁵NH₄NO₃ or NH ₄ ¹⁵ NO₃. The average N₂O emission rate was 24.2 ng N?kg^?1 h^?1 in WF soil, but it ranged from 69.6 to 507 ng N?kg^?1 h^?1 in VF soils. Autotrophic nitrification, heterotrophic nitrification and denitrification accounted for 0.3–31.4%, 25.4–54.4% and 22.5–57.7% of the N₂O emissions, respectively. When vegetable soils were moderately acidified (pH, 6.2 to ?≥?5.7), the increased N₂O emissions resulted from the increase of both the gross autotrophic and heterotrophic nitrification rates and the N₂O product ratio of autotrophic nitrification. However, once severe acidification occurred (as in VF4, pH?≤?4.3) and salt stress increased, both autotrophic and heterotrophic nitrification rates were inhibited to levels similar to those of WF soil. The enhanced N₂O product ratios of heterotrophic nitrification (4.84‰), autotrophic nitrification (0.93‰) and denitrification processes were the most important factors explaining high N₂O emission in VF4 soil. Data from this study showed that various soil conditions (e.g., soil salinity and concentration of NO ₃ ^- or NH ₄ ⁺ ) could also significantly affect the sources and rates of N₂O emission.     

The stabilisation potential of individual and mixed assemblages of natural bacteria and microalgae

It is recognized that microorganisms inhabiting natural sediments significantly mediate the erosive response of the bed ("ecosystem engineers") through the secretion of naturally adhesive organic material (EPS: extracellular polymeric substances). However, little is known about the individual engineering capability of the main biofilm components (heterotrophic bacteria and autotrophic microalgae) in terms of their individual contribution to the EPS pool and their relative functional contribution to substratum stabilisation. This paper investigates the engineering effects on a non-cohesive test bed as the surface was colonised by natural benthic assemblages (prokaryotic, eukaryotic and mixed cultures) of bacteria and microalgae. MagPI (Magnetic Particle Induction) and CSM (Cohesive Strength Meter) respectively determined the adhesive capacity and the cohesive strength of the culture surface. Stabilisation was significantly higher for the bacterial assemblages (up to a factor of 2) than for axenic microalgal assemblages. The EPS concentration and the EPS composition (carbohydrates and proteins) were both important in determining stabilisation. The peak of engineering effect was significantly greater in the mixed assemblage as compared to the bacterial (x 1.2) and axenic diatom (x 1.7) cultures. The possibility of synergistic effects between the bacterial and algal cultures in terms of stability was examined and rejected although the concentration of EPS did show a synergistic elevation in mixed culture. The rapid development and overall stabilisation potential of the various assemblages was impressive (x 7.5 and ×9.5, for MagPI and CSM, respectively, as compared to controls). We confirmed the important role of heterotrophic bacteria in "biostabilisation" and highlighted the interactions between autotrophic and heterotrophic biofilm consortia. This information contributes to the conceptual understanding of the microbial sediment engineering that represents an important ecosystem function and service in aquatic habitats.     

Influence of Algal Biomass on Extracellular Enzyme Activity in River Biofilms

Abstract Relationships between biofilm structural components (algal and bacterial biomass) and the activities of some extracellular enzymes that contribute to the ability to degrade organic matter) were explored for six Atlantic and three Mediterranean streams and rivers. The biofilms in these fluvial systems accounted for a wide range of bacterial and algal biomass and colonized the most common benthic habitats. Ratio of bacteria/algae biomass was lower in Atlantic than in Mediterranean streams, but enzymatic activities (β-glucosidase, β-xylosidase, phosphatase) were in general greater in the Mediterranean stream biofilms. Climatic characteristics (especially temperature) may explain the differences in enzymatic activities between biofilms of similar structure but different flow regime. The ratio β-xylosidase: β-glucosidase was similar (around 0.5) for all streams and substrata considered, showing that there is a general higher utilization of cellobiosic than xylobiosic molecules in fluvial systems. In general, highly heterotrophic biofilms showed lower extracellular enzymatic activities than more autotrophic biofilms. Maximum enzymatic activity is achieved when the algal biomass is two- to threefold higher than the bacterial biomass. The relevance of algal biomass on the heterotrophic ability of biofilms may be related to the physical proximity between the two, but also to the high proportion of polymeric carbohydrates included in algal exudates and lysis products, whose use is enzyme-mediated. Received: January 2000; Accepted: April 2000; Online Publication: 18 July 2000     

Role of heterotrophic nitrifiers and aerobic denitrifiers in simultaneous nitrification and denitrification process: a nonconventional nitrogen removal pathway in wastewater treatment

Bacterial species capable of performing both nitrification and denitrification in a single vessel under similar conditions have gained significance in the wastewater treatment scenario considering their unique character of performing the above reactions under heterotrophic and aerobic conditions respectively. Such a novel strategy often referred to as simultaneous nitrification and denitrification (SND) has a tremendous potential in dealing with various wastewaters having low C : N content, considering that the process needs very little or no external carbon source and oxygen supply thus adding to its cost-effective and environmentally friendly nature. Though like other micro-organisms, heterotrophic nitrifiers and aerobic denitrifiers convert inorganic or organic nitrogen-containing substances into harmless dinitrogen gas in the wastewater, their ecophysiological role in the global nitrogen cycle is still not fully understood. Attempts to highlight the role played by the heterotrophic nitrifiers and aerobic denitrifiers in dealing with nitrogen pollution under various environmental operating conditions will help in developing a mechanistic understanding of the SND process to address the issues faced by the traditional methods of aerobic autotrophic nitrification–anaerobic heterotrophic denitrification.     

Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin

A novel method using excision and fixation in Nanoplast, a hydrophilic embedding resin, allows confocal imaging of natural microbial communities and their extracellular polymeric secretions (EPS) while in situ. Prestaining with fluorescent probes permits the observation of specific cellular and extracellular components. Marine stromatolite sediments were examined using this method. Optical sectioning using confocal laser scanning microscopy (CLSM) permitted high-resolution imaging through sediments. Delicate arrangements of the EPS that are associated with sedimentary microbial biofilms were imaged using a fluorescein isothiocyanate (FITC)-labeled lectin (concanavalin-A) probe. Close microspatial associations of heterotrophic bacteria cells and autotrophic cyanobacteria cells were also observed. The nanoplast resin produces no detectable autofluorescence. Further coupling of multi-photon scanning laser microscopy (2P-LSM) with a conventional single photon CLSM allowed concurrent imaging of DAPI-labeled microbial cells, FITC-labeled EPS and autofluorescent carbonate sand grains. The multi-photon infrared laser permits deep (approximately 1 mm) penetration of samples and the excitation of DAPI, which normally requires UV-excitation with minimal disturbance to samples. The unique combination of Nanoplast with fluorescent probes, CLSM and 2P-LSM allows for the preservation and imaging of natural microbial communities in their in situ state, a method easily adapted for examinations of other microbial systems.     

Formation of nitrifying biofilms on small suspended particles in airlift reactors

For a stable and reliable operation of a BAS-reactor a high, active biomass concentration is required with mainly biofilm-covered carriers. The effect of reactor conditions on the formation of nitrifying biofilms in BAS-reactors was investigated in this article. A start-up strategy to obtain predominantly biofilm-covered carriers, based on the balancing of detachment and a biomass production per carrier surface area, proved tp be very successful. The amount of biomass and the fraction of covered carrier were high and development of nitrification activity was fast, leading to a volumetric conversion of 5 kg(N) . m(-3) . d(-1) at a hydraulic retention time of 1h. A 1-week, continuous inoculation with suspended purely nitrifying microorganisms resulted in a swift start-up compared with batch addition of a small number of biofilms with some nitrification activity. The development of nitrifying biofilms was very similar to the formation of heterotrophic biofilms. In contrast to heterotrophic bio-films, the diameter of nitrifying biofilms increased during start-up. The detachment rate from nitrifying biofilms decreased with lower concentrations of bare carrier, in a fashion comparable with heterotrophic biofilms, but the nitrifying biofilms were much more robust and resistant. Standard diffusion theory combined with reaction kinetics are capable of predicting the activity and conversion of biofilms on small suspended particles. (c) 1995 John Wiley & Sons Inc.     

A metaproteomic assessment of winter and summer bacterioplankton from Antarctic Peninsula coastal surface waters

A metaproteomic survey of surface coastal waters near Palmer Station on the Antarctic Peninsula, West Antarctica, was performed, revealing marked differences in the functional capacity of summer and winter communities of bacterioplankton. Proteins from Flavobacteria were more abundant in the summer metaproteome, whereas winter was characterized by proteins from ammonia-oxidizing Marine Group I Crenarchaeota. Proteins prevalent in both seasons were from SAR11 and Rhodobacterales clades of Alphaproteobacteria, as well as many lineages of Gammaproteobacteria. The metaproteome data were used to elucidate the main metabolic and energy generation pathways and transport processes occurring at the microbial level in each season. In summer, autotrophic carbon assimilation appears to be driven by oxygenic photoautotrophy, consistent with high light availability and intensity. In contrast, during the dark polar winter, the metaproteome supported the occurrence of chemolithoautotrophy via the 3-hydroxypropionate/4-hydroxybutyrate cycle and the reverse tricarboxylic acid cycle of ammonia-oxidizing archaea and nitrite-oxidizing bacteria, respectively. Proteins involved in nitrification were also detected in the metaproteome. Taurine appears to be an important source of carbon and nitrogen for heterotrophs (especially SAR11), with transporters and enzymes for taurine uptake and degradation abundant in the metaproteome. Divergent heterotrophic strategies for Alphaproteobacteria and Flavobacteria were indicated by the metaproteome data, with Alphaproteobacteria capturing (by high-affinity transport) and processing labile solutes, and Flavobacteria expressing outer membrane receptors for particle adhesion to facilitate the exploitation of non-labile substrates. TonB-dependent receptors from Gammaproteobacteria and Flavobacteria (particularly in summer) were abundant, indicating that scavenging of substrates was likely an important strategy for these clades of Southern Ocean bacteria. This study provides the first insight into differences in functional processes occurring between summer and winter microbial communities in coastal Antarctic waters, and particularly highlights the important role that ‘dark'' carbon fixation has in winter.     

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.