Production of medium-chain-length PHA in octanoate-fed enrichments dominated by Sphaerotilus sp.

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by using microbial enrichments is a promising but largely unexplored approach to obtain elastomeric biomaterials from secondary resources. In this study, several enrichment strategies were tested to select a community with a high mcl-PHA storage capacity when feeding octanoate. On the basis of analysis of the metabolic pathways, the hypothesis was formulated that mcl-PHA production is more favorable under oxygen-limited conditions than short-chain-length PHA (scl-PHA). This hypothesis was confirmed by bioreactor experiments showing that oxygen limitation during the PHA accumulation experiments resulted in a higher fraction of mcl-PHA over scl-PHA (i.e., a PHA content of 76 wt% with an mcl fraction of 0.79 with oxygen limitation, compared to a PHA content of 72 wt% with an mcl-fraction of 0.62 without oxygen limitation). Physicochemical analysis revealed that the extracted PHA could be separated efficiently into a hydroxybutyrate-rich fraction with a higher Mw and a hydroxyhexanoate/hydroxyoctanoate-rich fraction with a lower Mw . The ratio between the two fractions could be adjusted by changing the environmental conditions, such as oxygen availability and pH. Almost all enrichments were dominated by Sphaerotilus sp. This is the first scientific report that links this genus to mcl-PHA production, demonstrating that microbial enrichments can be a powerful tool to explore mcl-PHA biodiversity and to discover novel industrially relevant strains.

Moderate oxygen depletion as a factor favouring the filamentous growth of Sphaerotilus natans.

Sphaerotilus natans is a neutrophilic iron-related sheath-forming filamentous microorganism that presents dual morphotype: single cells and ensheathed cells forming filaments. As S. natans has been proposed as a sorbent for inorganic pollutants and it is occasionally involved in bulking episodes, elucidating factors affecting its filamentous growth is of crucial interest. The purpose of this work was to evaluate the effect of dissolved oxygen (DO) as a factor affecting S. natans filamentation from single cells. A method to quantify S. natans in its filamentous and single-cell morphotypes, based on a differential filtration procedure coupled with quantitative real-time PCR, was developed here. Scanning Electron Microscopy was used to validate the filtration step. Under actively aerated conditions (DO maintained at 7.6 ± 0.1 mg l(-1)), S. natans grew mainly as single cells throughout the experiment, while a depletion in DO concentration (to ~3 mg l(-1)) induced its filamentous growth. Indeed, when oxygen was reduced the proportion of single cells diminished from 83.3 ± 5.9 to 14.3 ± 3.4% while the filaments increased from 16.7 ± 5.9 to 85.7 ± 3.4%. Our results suggest that oxygen plays a key role in S. natans filamentation and contribute to better understanding of the filamentous proliferation of this bacterium. In addition, the proposed method will be helpful to evaluate other factors favouring filamentous growth.

Isolation of Sphaerotilus-Leptothrix strains from iron bacteria communities in Tierra del Fuego wetlands.

Sheath-forming iron- and manganese-depositing bacteria belonging to the Sphaerotilus-Leptothrix group (SLG) are widespread in natural and artificial water systems. Known requirements for their growth include the presence of organic substrates and molecular oxygen. High concentrations of reduced iron or manganese, although not necessary for most species, make their growth a noticeable phenomenon. Such microbial communities have been studied mostly in the Northern Hemisphere. Here, we present descriptions of diverse ochre-depositing microbial communities in Tierra del Fuego, Argentina, using a combined approach of microscopical examination, clone library construction and cultivation focused on SLG bacteria. To date, only few SLG type strains are available. The present work increases the number and diversity of cultivated SLG bacteria by obtaining isolates from biofilms and sediment samples of wetlands in Tierra del Fuego. Thirty isolates were selected based on morphological features such as sheath formation and iron/manganese deposition. Five operational taxonomic units (OTUs) were deduced. Sequencing of 16S rRNA genes showed that one OTU is identical to the Leptothrix mobilis Feox-1(T) -sequence while the four remaining OTUs show similarity values related to previously described type strains. Similarity values ranged from 96.5% to 98.8%, indicating possible new species and subspecies.

Sphaerotilus natans encrusted with nanoball-shaped Fe(III) oxide minerals formed by nitrate-reducing mixotrophic Fe(II) oxidation.

Ferrous iron has been known to function as an electron source for iron-oxidizing microorganisms in both anoxic and oxic environments. A diversity of bacteria has been known to oxidize both soluble and solid-phase Fe(II) forms coupled to the reduction of nitrate. Here, we show for the first time Fe(II) oxidation by Sphaerotilus natans strain DSM 6575(T) under mixotrophic condition. Sphaerotilus natans has been known to form a sheath structure enclosing long chains of rod-shaped cells, resulting in a thick biofilm formation under oxic conditions. Here, we also demonstrate that strain DSM 6575(T) grows mixotrophically with pyruvate, Fe(II) as electron donors and nitrate as an electron acceptor and single cells of strain DSM 6575(T) are dominant under anoxic conditions. Furthermore, strain DSM 6575(T) forms nanoball-shaped amorphous Fe(III) oxide minerals encrusting on the cell surfaces through the mixotrophic iron oxidation reaction under anoxic conditions. We propose that cell encrustation results from the indirect Fe(II) oxidation by biogenic nitrite during nitrate reduction and that causes the bacterial morphological change to individual rod-shaped single cells from filamentous sheath structures. This study extends the group of existing microorganisms capable of mixotrophic Fe(II) oxidation by a new strain, S. natans strain DSM 6575(T) , and could contribute to biogeochemical cycles of Fe and N in the environment.

Conformational analysis of an extracellular polysaccharide produced by Sphaerotilus natans.

Sphaerotilus natans is a filamentous sheath-forming bacterium, commonly found in bulking activated sludge. The bulky nature of this bacterium is caused by an extracellular polysaccharide (EPS). EPS is a linear acidic polysaccharide with the following chemical structure: [ → 4)-α-D-Glcp-(1 → 2)-ß-D-GlcpA-(1 → 2)-α-L-Rhap-(1 → 3)-ß-L-Rhap-(1 → ](n). (1)H-(1)H and (1)H-(13)C correlation nuclear magnetic resonance (NMR) experiments were performed to acquire nuclear Overhauser effect signals, which were used for conformational elucidation. Molecular mechanics calculations were performed on each disaccharide unit of the EPS building blocks. On the basis of the results of the calculations, the conformation of a pentasaccharide fragment was estimated. After confirmation of the coincidence between the NMR data and the predicted conformation of the pentasaccharide fragment, the conformation of a heptadecasaccharide fragment was estimated using the same procedure. The heptadecasaccharide was found to form a (12/1) helix and take locally folded chain, which is attributed to a triangular arrangement formed by a series of residues, α-D-Glcp-(1 → 2)-ß-D-GlcpA-(1 → 2)-α-L-Rhap. This arrangement is caused by the peculiar consecutive (1 → 2) linkages and reinforced by a hydrogen bond between the α-glucosyl and α-rhamnosyl residues. Considering the steric hindrance due to this triangular arrangement, EPS molecules are not supposed to form double helix in an aqueous environment. We propose the name 'sphaeran' to refer to this unique chemical structure and properties of EPS.

Biofilm formation and microbial community analysis of the simulated river bioreactor for contaminated source water remediation.

BACKGROUND, AIM, AND SCOPE: The start-up pattern of biofilm remediation system affects the biofilm characteristics and operating performances. The objective of this study was to evaluate the performances of the contaminated source water remediation systems with different start-up patterns in view of the pollutants removal performances and microbial community succession. METHODS: The operating performances of four lab-scale simulated river biofilm reactors were examined which employed different start-up methods (natural enrichment and artificial enhancement via discharging sediment with influent velocity gradient increase) and different bio-fillers (Elastic filler and AquaMats® ecobase). At the same time, the microbial communities of the bioreactors in different phases were analyzed by polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. RESULTS AND DISCUSSION: The pollutants removal performances became stable in the four reactors after 2 months' operation, with ammonia nitrogen and permanganate index (COD(Mn)) removal efficiencies of 84.41-94.21% and 69.66-76.60%, respectively. The biomass of mature biofilm was higher in the bioreactors by artificial enhancement than that by natural enrichment. Microbial community analysis indicated that elastic filler could enrich mature biofilm faster than AquaMats®. The heterotrophic bacteria diversity of biofilm decreased by artificial enhancement, which favored the ammonia-oxidizing bacteria (AOB) developing on the bio-fillers. Furthermore, Nitrosomonas- and Nitrosospira-like AOB coexisted in the biofilm, and Pseudomonas sp., Sphaerotilus sp., Janthinobacterium sp., Corynebacterium aurimucosum were dominant in the oligotrophic niche. CONCLUSION: Artificial enhancement via the combination of sediment discharging and influent velocity gradient increasing could enhance the biofilm formation and autotrophic AOB enrichment in oligotrophic niche.

Taxonomic investigation of representatives of the genus Sphaerotilus: descriptions of Sphaerotilus montanus sp. nov., Sphaerotilus hippei sp. nov., Sphaerotilus natans subsp. natans subsp. nov. and Sphaerotilus natans subsp. sulfidivorans subsp. nov., and an emended description of the genus Sphaerotilus.

Seven strains of the genus Sphaerotilus were obtained from natural thermal sulfide (strains D-501(T), D-502, D-504, D-505 and D-507) and low-temperature ferrous (strain HS(T)) springs and from an activated sludge system (strain D-380). These Sphaerotilus isolates and strains of Sphaerotilus natans obtained from the DSMZ (S. natans DSM 6575(T), DSM 565 and DSM 566) were studied using a polyphasic taxonomic approach. All strains had Q-8 as the major quinone and C(16 : 1)ω7, C(16 : 0) and C(18 : 1)ω7 as the major fatty acids. The DNA-DNA hybridization results and 16S rRNA, hsp60 and gyrB gene sequencing experiments showed that isolates D-501(T), D-502, D-504, D-505, D-507 and D-380 were closely related to the type strain of S. natans DSM 6575(T). However, strains D-501(T), D-502, D-504, D-505 and D-507 significantly differed from the heterotrophic strain S. natans DSM 6575(T) by their capability for lithotrophic growth with reduced sulfur compounds as an electron donor for energy conservation and some other phenotypic features. For this reason, strains D-501(T), D-502, D-504, D-505 and D-507 merit a separate taxonomic classification at the subspecies level. The name Sphaerotilus natans subsp. sulfidivorans subsp. nov. (type strain D-501(T) = DSM 22545(T) = VKM B-2573(T)) is proposed. The subspecies Sphaerotilus natans subsp. natans subsp. nov. is automatically created as a result of this proposal. Strain D-380 was phenotypically closely related to S. natans DSM 6575(T). Strains D-380 and S. natans DSM 6575(T) were assigned to the subspecies Sphaerotilus natans subsp. natans subsp. nov. (type strain DSM 6575(T) = ATCC 13338(T)). The 16S rRNA, hsp60 and gyrB gene sequences obtained for strains HS(T) and DSM 565 showed very low sequence similarity values of 97.3 %, 89.7 % and 88.4 %, respectively, with S. natans DSM 6575(T). Strain HS(T) shared 99 % DNA-DNA relatedness with strain.

[Ecophysiology of lithotrophic sulfur-oxidizing Sphaerotilus species from sulfide springs in the Northern Caucasus].

Six strains of sulfur-oxidizing bacteria of the known organotrophic species Sphaerotilus natans were isolated from two North Caucasian sulfide springs. Similar to known colorless sulfur bacteria, all the strains accumulated elemental sulfur when grown in media with sulfide. Unlike previously isolated S. natans strains, new isolates had higher temperature growth optima (33-37 degrees C) and variable metabolism. All the strains were capable of organotrophic, lithoheterotrophic, and mixotrophic growth with sulfur compounds as electron donors for energy metabolism. Variable metabolism of new Sphaerotilus isolates is a highly important adaptation mechanism which facilitates extension of their geographic range and supports their mass development in new habitats, e.g. sulfide springs. Within the cluster of new isolates, the physiological heterogeneity was shown to result from the inducible nature of the enzymes of oxidative sulfur metabolism and from their resistance to aerobic cultivation.

About the performance of Sphaerotilus natans to reduce hexavalent chromium in batch and continuous reactors.

The hexavalent chromium biological reduction constitutes a safe and economical detoxification procedure of wastewaters containing Cr(VI). However, little research has been done to evaluate Cr(VI) tolerance and reduction capacity of microbial cultures under different growth conditions. The aims of this work were (a) to evaluate the capacity of Sphaerotilus natans to reduce Cr(VI) to Cr(III) in a continuous system limited in carbon and energy source or in nitrogen source, (b) to evaluate the toxic effect of Cr(VI) on this microorganism, (c) to carry out a complete analysis of Cr(VI) reduction by S. natans not only in continuous regime but also in batch system, and (d) to model the obtained results mathematically. S. natans exhibited great resistance to Cr(VI) (19-78 mg l(-1)) and optimal growth in continuous and batch systems using a mineral medium supplemented only with citric acid as organic substrate. In carbon- and energy-limited continuous systems, a maximum percentual decrease in Cr(VI) by 13% was reached for low influent Cr(VI) concentration (4.3-5.32 mg Cr(VI)l(-1)); the efficiency of the process did not notoriously increase as the length of cellular residence time was increased from 4.16 to 50h. A nitrogen-limited continuous operation with a cellular residence time of 28.5h resulted in a Cr(VI) decrease of approximately 26-32%. In batch system, a mathematical model allowed to predict the Cr(VI) concentration as a function of time and the ratio between the initial Cr(VI) concentration and that of the biomass. High concentrations of initial Cr(VI) and biomass produced the highest performance of the process of Cr(VI) reduction reached in batch system, aspects which should be considered in detoxification strategies of wastewaters.

Reduction of hexavalent chromium by Sphaerotilus natans a filamentous micro-organism present in activated sludges.

Wastewaters produced by various industries may contain undesirable amounts of hexavalent chromium (Cr(VI)), as chromate and dichromate, a hazardous metal affecting flora and animals of aquatic ecosystems as well as human health. One removal strategy comprises the microbial reduction of Cr(VI) to Cr(III), a less soluble chemical species that is less toxic than Cr(VI). In this work, the ability to reduce Cr(VI) of Sphaerotilus natans, a filamentous bacterium usually found in activated sludge systems, was evaluated. In aerobic conditions, S. natans was able to efficiently reduce Cr(VI) to Cr(III) from dichromate solutions ranging between 4.5 and 80 mg Cr(VI)l(-1) in the presence of a carbonaceous source. A simultaneous evaluation of the microbial respiratory activity inhibition was also carried out to analyze the toxic effect of Cr(VI). Cr(VI) reduction by S. natans was mathematically modeled; chromium(VI) reduction rate depended on both Cr(VI) concentration and active biomass concentration. Although it is known that S. natans removes heavy metal cations such as Cr(III) by biosorption, the ability of this micro-organism to reduce Cr(VI), which behaves as an oxyanion in aqueous solutions, is a novel finding. The distinctive capacity to reduce Cr(VI) to Cr(III) than remain soluble or precipitated becomes S. natans a potential micro-organism to decontaminate wastewaters.

Structural analysis of the fundamental polymer of the sheath constructed by Sphaerotilus natans.

The sheath of Sphaerotilus natans is composed of cysteine-rich peptide and polysaccharide moieties. The polysaccharide was prepared by treating the sheath with hydrazine, and was determined to be a mucopolysaccharide containing beta-D-GlcA, beta-D-Glc, alpha-D-GalN, and beta-D-GalN. To elucidate the structure of the peptide, the sheath was labeled with a thiol-selective fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Enantiomeric determination of the S-derivatized Cys in the fluorescent sheath suggested that it contained L-Cys mainly. Fluorescent cysteinylglycine was detected in the partial acid hydrolysate of the fluorescent sheath. The sheath-degrading enzyme secreted by Paenibacillus koleovorans produced a fluorescent disaccharide-dipeptide composed of GalN, Gly, and N-acetylated Cys from the fluorescent sheath. The disaccharide and dipeptide moieties were found to be connected by an amide bond. Based on these results, the sheath was deduced to be formed by association of a mucopolysaccharide modified with N-acetyl-L-cysteinylglycine.

[Removing Cr(VI) by composite biosorbent of nano FeO4/Sphaerotilus natans].

The process of biosorption and the adsorbing mechanisms of Fe3 O4/Sphaerotilus natans to Cr(VI) were studied. The experiment results showed that pH value was main influencing factors on adsorption to Cr(VI). In the range of pH 2 to 3, the adsorbance of the composite biosorbent to Cr(VI) is 0.0217 mmol/g. The regeneration efficiency is 90% when the composite biosorbent was regenerated by HCl solution. --CONH2-- and --NH-- were principal activated groups of composite biosorbent. Cr(VI) can primarily combine with the surface of Fe3O4/Sphaerotilus natans by electrostatic attraction.

[Study on the disposal process for removing heavy metal ions from wastewater by composite biosorbent of nano Fe3O4/Sphaerotilus natans].

Composite biosorbent nano Fe3O4/Sphaerotilus natans was prepared when nano Fe3O4 was used as carrier, then the composite biosorbent characterized by infrared instrument and investigated on adsorbing heavy metal ions from wastewater. Infrared spectrum analysis shows that--CONH--and--OH are principal activated groups of composite biosorbent. The experiment of adsorption shows that biomass concentration and flow velocity are main influencing factors. The composite biosorbent has best effect on the adsorption of Cu2+ under the conditions of biomass 1.5 g/L (Sphaerotilus natans/Fe3O4 = 3:2), flow velocity 0.96 L/h and initial concentration of Cu2+ was less than 20 mg/L. Composite biosorbent can be used for more 10 times when biosorbent was regenerated by HCl solution, and HCl solution can be used 3 times repeatedly. The selective sequence was Ph2+ > Cu2+ > Zn2+ > Cd2+.

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA.

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.

Paenibacillus hodogayensis sp. nov., capable of degrading the polysaccharide produced by Sphaerotilus natans.

Sphaerotilus natans is a sheathed bacterium often found in activated sludge that has a bulking problem. A bacterial strain that is able to degrade the extracellular polysaccharide produced by S. natans was isolated. The isolate was a spore-forming, aerobic, rod-shaped bacterium. The Gram reaction was variable or negative. The optimum growth temperature was 30 degrees C and the optimum pH was 8. The G+C content of the DNA was 55 mol%. The major cellular fatty acid and respiratory quinone were anteiso-C(15 : 0) and MK-7, respectively. Phylogenetic analysis based on the 16S rRNA gene indicated that the isolate was a member of the genus Paenibacillus. The nearest relative, with a similarity of 94.2 %, was Paenibacillus koleovorans, a bacterium capable of degrading the sheath of S. natans. The phenotypic characteristics of the isolate were apparently different from those of related species in the genus Paenibacillus. It is proposed that the isolate be designated Paenibacillus hodogayensis sp. nov. The type strain is SG(T) (=JCM 12520(T)=KCTC 3919(T)).

[Research on Pb2 + removal from wastewater by Sphaerotilus natans].

As biosorbent used Sphaerotilus natans, influencing factors on removal of Pb2+ were investigated such as ratio of the biomass and lead, pH, temperature and time of adsorption. The results show that Sphaerotilus natans has good effect on adsorption of Pb2+. The process of adsorption reached equilibrium in 10 minutes. Temperature has no significant effect on the adsorption. Removal of Pb2+ approached to 100% under the condition of pH 5.5, 0.6 g/L and c0 < or =20 mg/L. The maximum adsorption amount was 2.1 mmol/g dry biomass . The adsorption was fitted with Freundlich isotherm adsorption equation under the concentration of Pb2+ 0-60 mg/L. HCI and EDTA can desorb the Pb2+ from the bacteria effectively.

Characterization of biofilms occurred in seepage groundwater contaminated with petroleum within an urban subway tunnel.

Biofilms occurring in seepage groundwater contaminated with petroleum in an urban subway drainage system were characterized. The development of biofilms was observed only in the sites where petroleum-contaminated groundwater had seeped or was seeping. Moreover, the conditions of the biofilms such as color and development extent were influenced by the amount of spilled petroleum: By increasing the amount of spilled petroleum, the amount of biofilms increased and its color whitened. It deteriorated and became dark-brown if the contaminated groundwater did not seep any more. These facts indicate that the biofilms can be used as a preliminary indicator to identify the locations of fuel contaminated sumps and seeps without a more detailed assessment such as instrumental analysis. The biofilms were capable of degrading petroleum at 15 degrees C, which is similar to the average temperature of the seepage groundwater. Filamentous bacteria, Sphaerotilus spp., were isolated from the biofilms. It is considered that these bacteria are responsible for the development of biofilms in the seepage groundwater contaminated with petroleum because they can secrete extracellular polymeric substances.