Sediment and Vegetation as Reservoirs of Vibrio vulnificus in the Tampa Bay Estuary and Gulf of Mexico

The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.     

Nutrient limitation of the primary production of phytoplankton in Lake Baikal

Nutrient limitation of the primary production of phytoplankton at some stations in southern and central Lake Baikal was studied by nutrient enrichment experiments in August 2002. Chlorophyll (Chl.) a concentrations ranged from 0.7 to 5.8μgl⁻¹. Inorganic nutrient concentrations were low: soluble reactive phosphorus ranged from 0.05 to 0.20μmoll⁻¹, ammonia from 0.21 to 0.41μmoll⁻¹, and nitrite plus nitrate from 0.33 to 0.37μmoll⁻¹. In the five enrichment experiments, phosphate spikes and phosphate plus nitrate spikes always stimulated primary production. Nitrate spikes also stimulated primary production in four of the experiments. Significant differences were detected between the controls and phosphate spikes and between the controls and phosphate plus nitrate spikes. Thus, the first limiting nutrient is thought to be phosphorus, but once phosphorus is supplied to the surface water, the limiting nutrient will quickly shift from phosphorus to nitrogen.     

Shallow-water habitats as sources of fallback foods for hominins

Underground storage organs (USOs) have been proposed as critical fallback foods for early hominins in savanna, but there has been little discussion as to which habitats would have been important sources of USOs. USOs consumed by hominins could have included both underwater and underground storage organs, i.e., from both aquatic and terrestrial habitats. Shallow aquatic habitats tend to offer high plant growth rates, high USO densities, and relatively continuous USO availability throughout the year. Baboons in the Okavango delta use aquatic USOs as a fallback food, and aquatic or semiaquatic USOs support high-density human populations in various parts of the world. As expected given fossilization requisites, the African early- to mid-Pleistocene shows an association of Homo and Paranthropus fossils with shallow-water and flooded habitats where high densities of plant-bearing USOs are likely to have occurred. Given that early hominins in the tropics lived in relatively dry habitats, while others occupied temperate latitudes, ripe, fleshy fruits of the type preferred by African apes would not normally have been available year round. We therefore suggest that water-associated USOs were likely to have been key fallback foods, and that dry-season access to aquatic habitats would have been an important predictor of hominin home range quality. This study differs from traditional savanna chimpanzee models of hominin origins by proposing that access to aquatic habitats was a necessary condition for adaptation to savanna habitats. It also raises the possibility that harvesting efficiency in shallow water promoted adaptations for habitual bipedality in early hominins. Am J Phys Anthropol 140:630–642, 2009. © 2009 Wiley-Liss, Inc.     

Concentrations of cadmium,lead, selenium,and zinc in human blood and seminal plasma

The concentrations of cadmium, lead, selenium, and zinc in blood and seminal plasma were determined in 76 Singapore males. Except for zinc, the concentrations were generally higher in blood than in seminal plasma (cadmium, 1.31 μg/L vs 0.61 μg/L; lead, 82.6 μg/L vs 12.4 μg/L, and selenium, 163.6 μg/L vs 71.5 μg/L). The mean concentration of zinc in seminal plasma was more than 30 times higher than in blood (202 mg/L vs 6.2 mg/L). Significant positive correlations were found between the concentrations in blood and seminal plasma for the two essential trace elements: selenium (r=0.45,p<0.001) and zinc (r=0.25,p<0.05). However, no relationships were found between the concentrations in blood and seminal plasma for two toxic metals (cadmium and lead). Significant inverse correlations were observed between Cd and Zn (r=−0.40,p<0.01), and Pb and Se (r=−0.32,p<0.05) in blood, whereas significant positive correlations were noted between Cd and Se (r=0.45,p<0.01), Cd and Zn (r=0.35,p<0.05), and Se and Zn (r=0.57,p<0.001) in seminal plasma. The physiological significance of these relationships are also discussed in this paper.     

Eutrophication and endangered aquatic plants: an experimental study on <Emphasis Type="Italic">Baldellia</Emphasis> <Emphasis Type="Italic">ranunculoides</Emphasis> (L.) Parl. (Alismataceae)

Anthropogenic enrichment of aquatic ecosystems is an increasingly common phenomenon, resulting from human population growth and the intensification of industrial and agricultural developments. Eutrophication has been considered to be one of the main causes of the decline of many aquatic plants. These observations, however, have rarely been supported by data or explored in detail using an experimental approach. Our experimental study demonstrates, for the first time, a strong direct negative influence of eutrophication on the performance of the endangered aquatic plant Baldellia ranunculoides s.str. (L.) Parl. (Alismataceae). Both morphological and reproductive traits were significantly affected by this phenomenon. Plants growing on eutrophic substrates were much smaller, possessed fewer and smaller leaves, and their biomass was on average half that of plants growing on a mesotrophic substrate. Additionally, plants growing on eutrophic substrates produced fewer inflorescences and flowers, and they had a smaller number of achenes per capitulum. Consequently, they produced only 15% of the seeds produced by plants growing in non-eutrophic conditions. Thus, the chance of long-term subsistence of B. ranunculoides populations in strongly eutrophic habitats will be significantly reduced, and at the same time, the success of any new establishment of viable B. ranunculoides populations in such habitats will be highly restricted. Conservation efforts for B. ranunculoides, either of existing populations or in newly selected re-introduction sites, should start, therefore, with an assessment of the trophic level of those sites. On the other hand, due to its sensitivity to eutrophication, the presence of viable populations of B. ranunculoides can be used as an indicator of valuable aquatic habitats for oligotrophy-dependent organisms.     

Distribution of egg strands of perch (<Emphasis Type="Italic">Perca</Emphasis> <Emphasis Type="Italic">fluviatilis</Emphasis> L.) with respect to depth and spawning substrate

Much of the historical Everglades has been either lost or degraded as a result of human activities. Among the aquatic habitats that comprise the Everglades landscape mosaic, open-water sloughs support critical ecological functions and appear especially sensitive to both hydrologic and water-quality perturbations. We used a combination of remote sensing and on-the-ground sampling to document spatial changes in the extent and vegetative composition of sloughs along a phosphorus (P) gradient in the northern Everglades. Increasing levels of water and soil P were associated with a decline in slough coverage, loss of the abundant native periphyton community, and a shift in dominant macrophyte species. The characteristic slough macrophyte species Eleocharis cellulosa and Nymphaea odorata exhibited different sensitivities to P enrichment, but both species declined with enrichment as slough habitats were invaded by Typha domingensis, a species that is known to expand aggressively in response to enrichment. A limited amount of open-water habitat occurred in highly enriched areas, but these habitats were maintained largely as a result of airboat disturbance and did not contain characteristic slough vegetation. Many changes in slough coverage and composition occurred in areas where water and soil P concentrations were only marginally higher than background levels. Our findings support the need for Everglades hydrologic restoration efforts to adhere to strict water-quality standards for P to avoid further degradation of this key landscape feature. Handling editor: L. M. Bini     

Physiological integration helps a clonal macrophyte spread into competitive environments and coexist with other species

Physiological integration may help clonal macrophytes invade or escape from existing communities. No studies have tested the above hypothesis in aquatic plants. In an outdoor pond experiment, we subjected clonal fragments of the submerged macrophyte Vallisneria spiralis L. to heterogeneous environments in which V. spiralis spread from bare habitats towards vegetated habitats occupied by Myriophyllum spicatum L. or V. spiralis spread from vegetated habitats towards bare habitats. V. spiralis stolons between ramets in bare habitats and in vegetated habitats were either intact or severed. We investigated the habitat selection of V. spiralis by examining the allocation of biomass and ramets to heterogeneous habitats during its vegetative spread phase. Results showed that the stolon connection had different effects on the habitat selection of V. spiralis with regard to invasion and escape. When V. spiralis spread from bare to vegetated habitats, in comparison to severing the stolon, the stolon connection eventually facilitated a 49% increase in biomass and a 27% increase in number of ramets allocated to vegetated habitats. However, when V. spiralis spread from vegetated to bare habitats, biomass and ramets allocated to bare habitats were not significantly changed by the stolon connection (only a 5% increase in biomass and a 6% increase in number of ramets). These results indicate that clonal integration facilitated V. spiralis not to escape from but invade into vegetated habitats. The study provides evidence that physiological integration is important for survival and tolerance of ramets in competitively stressful environments and can help clonal macrophytes coexist with other species.     

The Pattern of Change in the Abundances of Specific Bacterioplankton Groups Is Consistent across Different Nutrient-Enriched Habitats in Crete

A common source of disturbance for coastal aquatic habitats is nutrient enrichment through anthropogenic activities. Although the water column bacterioplankton communities in these environments have been characterized in some cases, changes in α-diversity and/or the abundances of specific taxonomic groups across enriched habitats remain unclear. Here, we investigated the bacterial community changes at three different nutrient-enriched and adjacent undisturbed habitats along the north coast of Crete, Greece: a fish farm, a closed bay within a town with low water renewal rates, and a city port where the level of nutrient enrichment and the trophic status of the habitat were different. Even though changes in α-diversity were different at each site, we observed across the sites a common change pattern accounting for most of the community variation for five of the most abundant bacterial groups: a decrease in the abundance of the Pelagibacteraceae and SAR86 and an increase in the abundance of the Alteromonadaceae, Rhodobacteraceae, and Cryomorphaceae in the impacted sites. The abundances of the groups that increased and decreased in the impacted sites were significantly correlated (positively and negatively, respectively) with the total heterotrophic bacterial counts and the concentrations of dissolved organic carbon and/or dissolved nitrogen and chlorophyll α, indicating that the common change pattern was associated with nutrient enrichment. Our results provide an in situ indication concerning the association of specific bacterioplankton groups with nutrient enrichment. These groups could potentially be used as indicators for nutrient enrichment if the pattern is confirmed over a broader spatial and temporal scale by future studies.     

Aquatic polymers can drive pathogen transmission in coastal ecosystems

Gelatinous polymers including extracellular polymeric substances (EPSs) are fundamental to biophysical processes in aquatic habitats, including mediating aggregation processes and functioning as the matrix of biofilms. Yet insight into the impact of these sticky molecules on the environmental transmission of pathogens in the ocean is limited. We used the zoonotic parasite Toxoplasma gondii as a model to evaluate polymer-mediated mechanisms that promote transmission of terrestrially derived pathogens to marine fauna and humans. We show that transparent exopolymer particles, a particulate form of EPS, enhance T. gondii association with marine aggregates, material consumed by organisms otherwise unable to access micrometre-sized particles. Adhesion to EPS biofilms on macroalgae also captures T. gondii from the water, enabling uptake of pathogens by invertebrates that feed on kelp surfaces. We demonstrate the acquisition, concentration and retention of T. gondii by kelp-grazing snails, which can transmit T. gondii to threatened California sea otters. Results highlight novel mechanisms whereby aquatic polymers facilitate incorporation of pathogens into food webs via association with particle aggregates and biofilms. Identifying the critical role of invisible polymers in transmission of pathogens in the ocean represents a fundamental advance in understanding and mitigating the health impacts of coastal habitat pollution with contaminated runoff.     

Tissue nutrient concentrations in freshwater aquatic macrophytes: high inter-taxon differences and low phenotypic response to nutrient supply

1. The elemental composition and stoichiometry of aquatic plants has often been suggested to reflect the nutrient enrichment of aquatic habitats. However, the relationship is often weak. Moreover, uncertainties remain in the relevance of laboratory derived critical plant tissue nutrient concentrations to maximum yield or growth rates in the field.
2. Aquatic vascular plants and bryophytes, overlying water and sediment samples were collected to test whether freshwater aquatic macrophytes: (i) show tissue nutrient deficiencies when growing in oligotrophic freshwater habitats, and (ii) have strict homeostatic stoichiometry.
3. Plant nutrient concentrations were significantly related to total inorganic nitrogen (or nitrate), total dissolved phosphorus and sediment total phosphorus. However, these relationships were weak. Virtually all the variance in plant tissue nutrient concentrations, however, could be explained by species (taxon) identity.
4. Critical tissue nutrient concentrations for 95% maximum yield or 95% maximum growth rate in aquatic angiosperms, determined from laboratory bioassays, suggested that nutrients should not limit yield in wild aquatic macrophytes. However, there were a substantial number of samples where potential growth rate limitation was possible, particularly due to phosphorus.
5. Strict C : N : P stoichiometric ratios were found for both vascular plants and bryophytes, suggesting little scope for plants as indicators of nutrient enrichment, but provide robust stoichiometric data for studies on ecosystem metabolism and nutrient cycling.     

The mycorrhizal status of an emergent aquatic, Lythrum salicaria L., at different levels of phosphorus availability

 The relationship between nutrient availability and mycorrhizal status has been well studied for terrestrial plant species, but has been examined rarely in aquatic and emergent aquatic species. The purpose of this study was to determine the effect of phosphorus availability on the arbuscular mycorrhizal (AM) status of an emergent aquatic, Lythrum salicaria L. L. salicaria was grown in hydroponic sand culture at five phosphorus concentrations (0, 100, 1000, 10 000, and 47 500 μg PO₄/l nutrient solution) for 49 days with or without mycorrhizal inoculum obtained from wetland soil. Inoculated plants at the lowest three phosphorus concentrations were colonized by AM, whereas there was no colonization of plants grown at the highest two phosphorus concentrations. Colonization by AM fungi occurred in conjunction with symptoms of phosphorus deficiency in L. salicaria under experimental conditions: plants at the lowest three phosphorus concentrations had lower biomass and higher root: shoot weight ratios than plants at the highest two concentrations. However, total biomass and internal phosphorus concentration did not differ between inoculated and control plants. Further studies are needed under conditions more closely mimicking natural dynamics. Accepted: 7 August 1999     

The ecological niche of the consortium “Pelochromatium roseum”

A dense accumulation of the phototrophic consortium “Pelochromatium roseum” in a small, eutrophic, freshwater lake (Dagowsee, Brandenburg, Germany) was investigated. Within the chemocline, the number of epibionts of the consortia represented up to 19% of the total number of bacteria. Per “P. roseum” a mean value of 20 epibionts was determined. Similar to other aquatic habitats, consortia in the Dagowsee were found only at low light intensities (< 7 μmol quanta m^–2 s^–1) and low sulfide concentrations (0–100 μM). In dialysis cultures of “P. roseum”, bacterial cells remained in a stable association only when incubated at light intensities between 5 and 10 μmol quanta m^–2 s^–1. Intact consortia from natural samples had a buoyant density of 1046.8 kg m^–3, which was much higher than that of ambient chemocline water (995.8 kg m^–3). Under environmental conditions and without motility, this density difference would result in rapid sedimentation of consortia toward the lake bottom. Our results indicate that (1) consortia are adapted to a very narrow regime of light intensities and sulfide concentrations, (2) motility and tactic responses must be of ecological significance for the colonization of the free water column of lakes, and (3) phototrophic growth of consortia can be explained only by a cycling of sulfur species in the chemocline, possibly within the consortia themselves. Received: 27 May 1997 / Accepted: 16 September 1997     

Seasonal habitat use indicates that depth may mediate the potential for invasive round goby impacts in inland lakes

1. The round goby (Neogobius melanostomus) is among the fastest-spreading introduced aquatic species in North America and is radiating inland from the Great Lakes into freshwater ecosystems across the landscape. Predicting and managing the impacts of round gobies requires information on the factors influencing their distribution in habitats along the invasion front, yet this information is not available for many recently invaded ecosystems. We evaluated the seasonal habitat use and biomass of round gobies in an inland temperate lake to define the spatiotemporal scope of biological interactions at the leading edge of the round goby invasion.
2. Using novel statistical approaches, we combined hierarchical models that control for imperfect species detection with flexible smooth terms to describe non-linear relationships between round goby abundance and environmental gradients. Subsequently, we generated accurate detection-corrected estimates of the standing stock biomass of round gobies.
3. Our results show seasonally differentiated habitat niches, where suitable round goby habitat in summer months is restricted to shallow depths (<18.4 m) with a mixture of vegetative and mussel cover. We found high round goby biomass of 122 kg/ha in occupied habitats during the summer, with a total lake-wide biomass of 766,000 kg. In winter, round gobies migrate to deep offshore habitats and disperse, dramatically altering their scope for biological interactions with resident aquatic species across summer and winter seasons.
4. The results of this study indicate that the scope of biological interactions in inland lakes may be seasonally variable, with potential for high round goby biomass in shallow lakes or at the periphery of deep lakes in the summer months. Such shallow-water habitats may therefore present higher risk of ecological impacts from round gobies in invaded lentic ecosystems. As round gobies expand inland, consideration of seasonal habitat use will be an important factor in predicting the impacts of this pervasive invader.

     

Effects of anti-microtubule drugs onin vitro polymerization of tubulin from mung bean

Effects of anti-microbule drugs on tubulin polymerizationin vitro were investigated using purified mung bean (Vigna radiata) tubuli. Colchicine induced the formation of macrotubules at the relatively low concentration of 10 μM. and the appearance of corkscrew-like filaments from the ends of the macrotubules at concentrations of more than 100 μM. Vinblastine substantially inhibited polymerization at 1 μM and caused the formation of paracrystals at concentrations greater than 10 μM. Oryzalin inhibited polymerization at 1 μM partially and at 10 μM completely. Paracrystal formation was also induced by cremart at 10 μM, but these paracrystals appeared to be more rigid than those induced by vinblastine. Amiprophos methyl (APM), with a chemical configuration similar to cremart, substantially inhibited polymerization at 1 μM, but the formation of paracrystals was weak. Griseofulvin at 10 μMalso inhibited the polymerization of tubulin while at higher concentrations aggregates of helices were formed. Inhibition of polymerization by phenylcarbamate herbicides was more effective than that caused by benzimidazoylcarbamate fungicides. The effects of drugs onin vitro preformed (MTs) were also investigated. Colchicine and vinblastine showed identical effects to those on the polymerization process. Griseofulvin, cremart and APM induced only macrotubule formation while the other drugs tested had no major effects     

Survival ofLegionella pneumophila in the aquatic environment

Survival ofLegionella pneumophila SG 1 in seawater and river water was assessed using plate counts on buffered charcoal yeast extract agar amended with α-ketoglutarate (BCYEα) and [³H]thymidine-labeling. The [³H]thymidine-labeling method for assessing survival ofL. pneumophila in aquatic environments was compared with viable counts, direct fluorescent microscopy (DFA), and acridine orange direct counts (AODC). Protozoa were isolated from the samples employed in the study and identified by characteristic trophozite and cyst morphology. Selective filtration employing 2.0 μm Nucleopore filters was used to determine the effect of grazing on survival ofL. pneumophila in seawater and river water.Legionella viability as measured by plate counts (CFU/ml), declined to a greater extent than cell lysis, assessed by thymidine, DFA, and AODC counts, suggesting thatL. pneumophila survives in aquatic habitats to a greater extent than revealed through culturable counts.     

Growth and fruitbody formation ofGanoderma lucidum on media supplemented with vanadium, selenium and germanium

Responses of mycelia ofGanoderma lucidum to vanadium, selenium and germanium were examined over a wide range of concentrations (10–1, 120 μg/ml) in pure culture. Se and V were found to be highly toxic, but Ge was not toxic at the levels tested.Ganododerma lucidum cultivated on substrates of sawdust with V (30–80 μg/g) developed mature fruitbodies, but the bioaccumulation of V was quite low (2.5–7 μg/g in pileus, 12.5–21.5 μg/g in stipe and <1 μg/g in basidiospores). Se as Na₂SeO₄ labeled with⁷⁵Se was effectively taken up from substrates and accumulated in fruitbodies (mainly in pileus), then depleted by discharge of basidiospores. Ge as GeCl₄ labeled with⁷⁷Ge was easily uptaken and translocated into fruitbodies.     

Studies on moisture relationships of some aquatic and terrestrial algae

Summary Significant variations among aquatic and terrestrial algae (L. arboricola, Oedogonium sp. and V. hamata), in moisture and hydration percentage have been observed. Both percentages proved to be significantly higher in the aquatic forms.     

Aerobic cometabolic degradation of trichloroethene by methane and ammonia oxidizing microorganisms naturally associated with <Emphasis Type="Italic">Carex comosa</Emphasis> roots

The degradation potential of trichloroethene by the aerobic methane- and ammonia-oxidizing microorganisms naturally associated with wetland plant (Carex comosa) roots was examined in this study. In bench-scale microcosm experiments with washed (soil free) Carex comosa roots, the activity of root-associated methane- and ammonia-oxidizing microorganisms, which were naturally present on the root surface and/or embedded within the roots, was investigated. Significant methane and ammonia oxidation were observed reproducibly in batch reactors with washed roots incubated in growth media, where methane oxidation developed faster (2 weeks) compared to ammonia oxidation (4 weeks) in live microcosms. After enrichment, the methane oxidizers demonstrated their ability to degrade 150 μg l⁻¹ TCE effectively at 1.9 mg l⁻¹ of aqueous CH₄. In contrast, ammonia oxidizers showed a rapid and complete inhibition of ammonia oxidation with 150 μg l⁻¹ TCE at 20 mg l⁻¹ of NH₄ ⁺-N, which may be attributed to greater sensitivity of ammonia oxidizers to TCE or its degradation product. No such inhibitory effect of TCE degradation was detected on methane oxidation at the above experimental conditions. The results presented here suggest that microorganisms associated with wetland plant roots can assist in the natural attenuation of TCE in contaminated aquatic environments.     

Microbial conversion and in vitro and in vivo antifungal assessment of bioconverted docosahexaenoic acid (bDHA) used against agricultural plant pathogenic fungi

Microbial modification of polyunsaturated fatty acids can often lead to special changes in their structure and in biological potential. Therefore, the aim of this study was to develop potential antifungal agents through the microbial conversion of docosahexaenoic acid (DHA). Bioconverted oil extract of docosahexaenoic acid (bDHA), obtained from the microbial conversion of docosahexaenoic acid (DHA) by Pseudomonas aeruginosa PR3, was assessed for its in vitro and in vivo antifungal potential. Mycelial growth inhibition of test plant pathogens, such as Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum, was measured in vitro. bDHA (5 μl disc⁻¹) inhibited 55.30–65.90% fungal mycelium radial growth of all the tested plant pathogens. Minimum inhibitory concentrations (MICs) of bDHA against the tested plant pathogens were found in the range of 125–500 μg ml⁻¹. Also, bDHA had a strong detrimental effect on spore germination for all the tested plant pathogens. Further, three plant pathogenic fungi, namely C. capsici, F. oxysporum and P. capsici, were subjected to an in vivo antifungal screening. bDHA at higher concentrations revealed a promising antifungal effect in vivo as compared to the positive control oligochitosan. Furthermore, elaborative study of GC-MS analysis was conducted on bioconverted oil extract of DHA to identify the transformation products present in bDHA. The results of this study indicate that the oil extract of bDHA has potential value of industrial significance to control plant pathogenic fungi.     

Bioaugmentation and enhanced formation of microbial granules used in aerobic wastewater treatment

Microbial aggregates of an aerobic granular sludge can be used for the treatment of industrial or municipal wastewater, but their formation from a microbial activated sludge requires several weeks. Therefore, the aim of this research was the selection of microbial cultures to shorten the granule-forming period from several weeks to a few days. An enrichment culture with the ability to accelerate granulation was obtained by repeating the selection and batch cultivation of fast-settling microbial aggregates isolated from the aerobic granular sludge. Bacterial cultures of Klebsiella pneumoniae strain B and Pseudomonas veronii strain F, with self-aggregation indexes of 65 and 51%, respectively, and a coaggregation index of 58%, were isolated from the enrichment culture. A mixture of these strains with the activated sludge was used as an inoculum in an experimental sequencing batch reactor to start up an aerobic granulation process. Aerobic granules with a mean diameter of 446±76 μm were formed in an experiment after 8 days of cultivation, but microbial granules were absent in controls. Considering biosafety issues, K. pneumoniae strain B was excluded from further studies, but P. veronii strain F was selected for larger-scale testing.Stephen Tiong-Lee Tay Passed away on 27 July 2005.