Application of quantitative RT-PCR to determine the distribution of <Emphasis Type="Italic">Microthrix parvicella</Emphasis> in full-scale activated sludge treatment systems

Three wastewater treatment plants in South Africa were investigated to understand the phylogeny and distribution of Microthrix parvicella using real-time polymerase chain reaction (RT-PCR). The phylogenetic analysis of the 16S rRNA of M. parvicella revealed 98% to 100% homology of South African clones to M. parvicella reported in Genbank. The standard curves for RT-PCR showed R2 values greater than 0.99, accurate for quantification. The relative occurrence of M. parvicella 16S rRNA gene copies in the three wastewater treatment plants was in the range 0% to 3.97%. M. parvicella copies increased when the environmental temperature (≤20°C) and food/microorganism (F/M) ratio was low. The M. parvicella 16S rRNA copies could be positively correlated to the sludge volume index at low temperature. At higher temperature, there was a rapid reduction in M. parvicella population irrespective of other favorable factors, indicating the strong influence of temperature on filamentous proliferation. RT-PCR has potential applications in wastewater treatment plants to monitor sudden shift in the microbial population and assessing the plants efficacy.     

Change in surface properties of Microthrix parvicella upon addition of polyaluminium chloride as characterized by atomic force microscopy

The filamentous bacterium Microthrix parvicella causes severe separation and foaming problems at wastewater treatment plants (WWTPs). An effective control of the bacterium in activated sludge WWTPs can be accomplished by dosage with polyaluminium chloride (PAX-14). The purpose of this study was to investigate whether addition of PAX-14 affects surface properties such as the hydrophobicity of the bacterium and to study the exopolymers of M. parvicella that host surface-associated enzymes. To this end, force measurements by atomic force microscopy were carried out to measure the interactions between hydrophilic and hydrophobized tips and the bacterium surface. Addition of PAX-14 caused no changes in the hydrophobicity of the bacterium surface but the data indicate that it collapsed the polymeric layer likely due to electrostatic screening. It is concluded that the collapse of the polymeric layer may affect the transport of substrates (eg free fatty acids) to the bacterium and hence the competitiveness of M. parvicella compared to the other bacteria present in activated sludge.     

Aluminium toxicity and binding to Escherichia coli

The toxicity and binding of aluminium to Escherichia coli has been studied. Inhibition of growth by aluminium nitrate was markedly dependent on pH; growth in medium buffered to pH 5.4 was more sensitive to 0.9 mM or 2.25 mM aluminium than was growth at pH 6.6–6.8. In medium buffered with 2-(N-morpholino)ethanesulphonic acid (MES), aluminium toxicity was enhanced by omission of iron from the medium or by use of exponential phase starter cultures. Analysis of bound aluminium by atomic absorption spectroscopy showed that aluminium was bound intracellularly at one type of site with a K _m of 0.4 mM and a capacity of 0.13 mol (g dry wt)^-1. In contrast, binding of aluminium at the cell surface occurred at two or more sites with evidence of cooperativity. Addition of aluminium nitrate to a weakly buffered cell suspension caused acidification of the medium attributable to displacement of protons from cell surfaces by metal cations. It is concluded that aluminium toxicity is related to pH-dependent speciation [with Al(H₂O) ₆ ³⁺ probably being the active species] and chelation of aluminium in the medium. Aluminium transport to intracellular binding sites may involve Fe(III) transport pathways.     

Sludge bulking impact on relevant bacterial populations in a full-scale municipal wastewater treatment plant

We have investigated the changes of microbial community structures and the concomitant performance in two biological wastewater treatment systems (conventional and inverted A²/O processes) over a whole cycle of sludge bulking. A low level of filament abundance was observed during non-bulking period, with types 0092 and 0041 as the dominant filamentous bacteria. With the increase of the sludge volume index values from 76 (73) to 275 (300) mg/L, the filament abundance estimated by microscopic examination increased from 1 (few) to 5 (abundant), with Microthrix parvicella becoming the dominant filament bacteria. Sludge bulking resulted in a significant shift in bacterial compositions from Proteobacteria to Actinobacteria dominance, characterized by the significant presence of filamentous M. parvicella (from not detected to higher than 60% of clones) and decrease of the dominant Betaproteobacterial population (from higher than 40% to less than 1%). Important relevant bacterial populations including polyphosphate-accumulating organism (PAO, Candidatus Accumulibacter phosphatis), ammonia-oxidizing bacteria (AOB, Nitrosomonas), nitrite-oxidizing bacteria (NOB, Nitrospira) and denitrifying bacteria (Thauera) were absent under the serious bulking condition. Accumulation of nitrite and ammonia was observed during serious bulking, while the phosphorus removal performance was not decreased because M. parvicella could behave as a PAO.     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.     

Metal cation binding by the hyperthermophilic microorganism,Archaea Methanocaldococcus Jannaschii,and its effects on silicification

Abstract: A series of experiments was conducted to determine the capacity of an archaeal strain, Methanocaldococcus jannaschii, to bind metals and to study the effects of metal binding on the subsequent silicification of the microorganisms. The results showed that M. jannaschii can rapidly bind several metal cations (Fe³⁺, Ca²⁺, Pb²⁺, Zn²⁺, Cu²⁺). Considering the lack of silicification of this strain without metal binding, these experiments demonstrate that Fe(III) ion binding to the cell wall components was of fundamental importance for successful silicification and, especially, for the excellent preservation of the cell wall. This study brings new elements to the understanding of fossilization processes, showing that the positive effect of Fe(III) on silicification, already known for Bacteria, can also apply to Archaea and that this preliminary binding can be decisive for the subsequent fossilization of these organisms. Knowledge of these mechanisms can be helpful for the search and the identification of microfossils in both terrestrial and extraterrestrials rocks, and in particular on Mars.     

Geochemistry and microbiology of an impounded subterranean acidic water body at Mynydd Parys, Anglesey, Wales

Acidic, metal‐rich water that had accumulated within two abandoned, adjacent copper mines in north Wales was removed to prevent its possible catastrophic release. About 274 000 m³ of acidic (pH ～2.4) mine water was pumped out of the mines over a 14‐week period. Concentrations of dissolved species (iron, sulfate, aluminium, copper, manganese and zinc) increased as water at lower depths within the mines was accessed. The discharged water flowed through a small wetland area, reaching the sea about 3 km north of the mine site. Analysis of the water at three sampling stations revealed that there was very little removal of aluminium and most of the heavy metals present except (ferrous) iron, which was partially removed as a result of oxidation and hydrolysis of the resulting ferric iron. The dominant bacterium in the subterranean mine water, which was essentially devoid of oxygen, was the iron‐ and sulfur‐oxidizer Acidithiobacillus ferrooxidans. The microbial populations in the pumped mine water were monitored using combined cultivation‐dependent (isolation on solid media) and cultivation‐independent (terminal restriction fragment length polymorphism and clone library) techniques. Other bacteria detected in the mine water included other iron‐oxidizers (Leptospirillum spp., ‘Ferrimicrobium acidiphilum’ and Gallionella‐like organisms) and heterotrophic acidophiles (Acidiphilium, Acidisphaera and Acidobacterium). Archaeal clones were also detected; most of these were related to methanogens. Owing to the absence of an effective remediation strategy, an estimated 7.5 tonnes of copper, 3.1 tonnes of manganese, 14.8 tonnes of zinc and 15.3 tonnes of aluminium was discharged into the Irish Sea as a consequence of the dewatering of the mines.     

Isolation and Characterization of Holocellulose from Alfalfa

Holocellulose isolated from the aerial parts of alfalfa (Medicago sativa) contains a polysaccharide complex of cellulose and hemicelluloses, the major structural components of cell walls. Holocellulose is highly hydrophilic and has a dense biopolymer packing. The carboxylic groups of hemicelluloses and cellulose determines the ability of holocellulose to adsorb polyvalent metal cations.     

Characterization of filamentous microorganisms in rotating biological contactor biofilms of wastewater treatment plants

Filamentous microorganisms populations present in biofilms from three wastewater treatment plants with a rotating biological contactor system have been studied. Results showed that filaments are an integral component of the biofilms, and that they were made up by: Beggiatoa ssp., Haliscomenobacter hydrossis, Sphaerotilus natans, Thiothrix ssp., and the 021N, 0803, 0914, 0961, 1701, 1851, and 1863 Eikelboom's types. Beggiatoa ssp. was the most frequent microorganism followed by S. natans and type 021N. Microthrix parvicella, Gordona amarae (=Nocardia amarae), Nostocoida limicola (I, II, III), and 0041, 0092, 0211, 0411, 0581, 0675, 1702, and 1852 Eikelboom's types appear infrequently.     

Stable foams in activated sludge plants

A study of several full-scale activated sludge plants has confirmed that whilst stable foam formation is associated with a dominance of the sludge flora by Nocardia or Microthrix parvicella, the degree of dominance is not related to the foaming potential as measured by a novel ‘foam index’. The data also show the significance that the suspended solids concentration has on foaming.     

Malate secretion from the root system is an important reason for higher resistance of Miscanthus sacchariflorus to cadmium

Miscanthus is a vigorous perennial Gramineae genus grown throughout the world as a promising bioenergy crop and generally regarded as heavy metal tolerant due to its ability to absorb heavy metals. However, little is known about the mechanism for heavy metal tolerance in Miscanthus. In this study, two Miscanthus species (Miscanthus sacchariflorus and Miscanthus floridulus) exhibiting different cadmium (Cd) sensitivity were used to address the mechanisms of Cd tolerance. Under the same Cd stress, M. sacchariflorus showed higher Cd tolerance with better growth and lower Cd accumulation in both shoots and roots than M. floridulus. The malate (MA) content significantly increased in root exudates of M. sacchariflorus following Cd treatment while it was almost unchanged in M. floridulus. Cellular Cd analysis and flux data showed that exogenous MA application markedly restricted Cd influx and accumulation while an anion‐channel inhibitor (phenylglyoxal) effectively blocked Cd‐induced MA secretion and increased Cd influx in M. sacchariflorus, indicating that MA secretion could alleviate Cd toxicity by reducing Cd uptake. The genes of malate dehydrogenases (MsMDHs) and Al‐activated malate transporter 1 (MsALMT1) in M. sacchariflorus were highly upregulated under Cd stress, compared with that in M. floridulus. The results indicate that Cd‐induced MA synthesis and secretion efficiently alleviate Cd toxicity by reducing Cd influx in M. sacchariflorus.     

Stable limited filamentous bulking through keeping the competition between floc-formers and filaments in balance

Limited filamentous bulking (LFB) was proposed to save aeration energy consumption and enhance the capacity of filaments to degrade substrates with low concentrations in activated sludge systems. Operational parameters favorable for maintaining the LFB state were investigated in an anoxic-oxic reactor treating domestic wastewater. The experiments showed that the LFB state would deteriorate with sharply decreasing temperature, reducing substrate gradients or removing anoxic zones. The balance between filaments and floc-formers could be achieved by controlling dissolved oxygen and sludge loading rates to be in optimal ranges. Eikelboom Type 0041 and CandidatusMicrothrix parvicella were the filamentous bacteria responsible for the LFB state. However, the excess growth of Eikelboom Type 021N and Sphaerotilus natans were observed when serious bulking occurred under low substrate gradients. It was demonstrated that stable maintenance of LFB for energy saving was feasible by process control and optimization.     

From industrial sites to environmental applications with <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>

Cupriavidus metallidurans CH34 and related strains are adapted to metal contaminated environments. A strong resistance to environmental stressors and adaptation make it ideal strains for survival in decreasing biodiversity conditions and for bioaugmentation purposes in environmental applications. The soil bacterium C. metallidurans is able to grow chemolithoautotrophically on hydrogen and carbon dioxide allowing a strong resilience under conditions lacking organic matter. The biofilm growth on soil particles allows coping with starvation or bad conditions of pH, temperature and pollutants. Its genomic capacity of two megaplasmids encoding several heavy metal resistance operons allowed growth in heavy metal contaminated habitats. In addition its specific siderophores seem to play a role in heavy metal sequestration besides their role in the management of bioavailable iron. Efflux ATPases and RND systems pump the metal cations to the membrane surface where polysaccharides serve as heavy metal binding and nucleation sites for crystallisation of metal carbonates. These polysaccharides contribute also to flotation under specific conditions in a soil-heavy metals–bacteria suspension mixture. An inoculated moving bed sand filter was constructed to treat heavy metal contaminated water and to remove the metals in the form of biomass mixed with metal carbonates. A membrane based contactor allowed to use the bacteria as well in a versatile wastewater treatment system and to grow homogeneously formed heavy metal carbonates. Its behaviour toward heavy metal binding and flotation was combined in a biometal sludge reactor to extract and separate heavy metals from metal contaminated soils. Finally its metal-induced heavy metal resistance allowed constructing whole cell heavy metal biosensors which, after contact with contaminated soil, waste, solids, minerals and ashes, were induced in function of the bioavailable concentration (Cd, Zn, Cu, Cr, Co, Ni, Tl, Pb and Hg) in the solids and allowed to investigate the speciation of immobilization of those metals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3–1000 µ.M) induced a smooth inward current that was competitively inhibted by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 µM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (E_r) of -6.1 ± 0.5 mV In cerebellar mRNA-injected oocytes; E_r was nearly identical (-5.1 ± 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd⁺⁺ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Dominance of ‘Gallionella capsiferriformans’ and heavy metal association with Gallionella‐like stalks in metal‐rich pH 6 mine water discharge

Heavy metal‐contaminated, pH 6 mine water discharge created new streams and iron‐rich terraces at a creek bank in a former uranium‐mining area near Ronneburg, Germany. The transition from microoxic groundwater with ~5 mm Fe(II) to oxic surface water may provide a suitable habitat for microaerobic iron‐oxidizing bacteria (FeOB). In this study, we investigated the potential contribution of these FeOB to iron oxidation and metal retention in this high‐metal environment. We (i) identified and quantified FeOB in water and sediment at the outflow, terraces, and creek, (ii) studied the composition of biogenic iron oxides (Gallionella‐like twisted stalks) with scanning and transmission electron microscopy (SEM, TEM) as well as confocal laser scanning microscopy (CLSM), and (iii) examined the metal distribution in sediments. Using quantitative PCR, a very high abundance of FeOB was demonstrated at all sites over a 6‐month study period. Gallionella spp. clearly dominated the communities, accounting for up to 88% of Bacteria, with a minor contribution of other FeOB such as Sideroxydans spp. and ‘Ferrovum myxofaciens’. Classical 16S rRNA gene cloning showed that 96% of the Gallionella‐related sequences had ≥97% identity to the putatively metal‐tolerant ‘Gallionella capsiferriformans ES‐2’, in addition to known stalk formers such as Gallionella ferruginea and Gallionellaceae strain R‐1. Twisted stalks from glass slides incubated in water and sediment were composed of the Fe(III) oxyhydroxide ferrihydrite, as well as polysaccharides. SEM and scanning TEM‐energy‐dispersive X‐ray spectroscopy revealed that stalk material contained Cu and Sn, demonstrating the association of heavy metals with biogenic iron oxides and the potential for metal retention by these stalks. Sequential extraction of sediments suggested that Cu (52–61% of total sediment Cu) and other heavy metals were primarily bound to the iron oxide fractions. These results show the importance of ‘G. capsiferriformans’ and biogenic iron oxides in slightly acidic but highly metal‐contaminated freshwater environments.     

Effect of medium composition on the ultrastructure of Lactobacillus strains

The growth of some locally isolated Lactobacillus strains forming D(-) or L(+) lactic acid, Lactobacillus helveticus ATCC 15009 and Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was examined in different media. L. helveticus and Lactobacillus LBL strains formed atypical protoplast-like cells in LAPT medium, sensitive to SDS and proteinase. Specific morphological changes in the cell wall structure of these variants were revealed by transmission and scanning electron microscopy. The effect of glucose and various salts on their appearance was investigated. The prevalent role of metal cations, especially of Mg²⁺, was established.     

Metal and antibiotic resistance traits in Bradyrhizobium sp. (cajanus) isolated from soil receiving oil refinery wastewater

Forty isolates of Bradyrhizobium sp. (cajanus) were isolated from the nodules of pigeon pea plants grown in fields receiving petrochemical industrial wastewater for the past 12 years and characterized using standard methods. The heavy metal analysis of field soil and treated wastewater showed their presence in varying concentrations. All isolates showed resistance to one or more metals at concentrations >200 g/ml. Multiple metal resistance was a common phenomenon in these isolates. There was no correlation between extractable soil metal concentration and the ability of the isolates to tolerate metal salts in their growth medium as evidenced from their minimum inhibitory concentration (MIC). However, high incidence of metal resistance and the multiple nature of resistance might have been the result of continuous exposure of these strains to heavy metals in the treated wastewater of Mathura Oil Refinery. These strains were also found to be resistant to one or more of the 13 antibacterial drugs tested.     

Antifungal activity of aluminium‐containing salts against the development of carrot cavity spot and potato dry rot

As an alternative to the use of synthetic chemical fungicides to control plant disease, aluminium‐containing salts were evaluated for their effects on the mycelial growth of various fungal or fungus‐like pathogens and their ability to control carrot cavity spot (Pythium sulcatum) and potato dry rot (Fusarium sambucinum). Results showed that various aluminium‐containing salts provided strong inhibition of all the tested pathogens (Alternaria solani, Botrytis cinerea, F. sambucinum, P. sulcatum and Rhizopus stolonifer) with minimal inhibitory concentration of 1–10 mM. Aluminium chloride and aluminium sulphate were generally the most effective, inhibiting mycelial growth of pathogens by as much as 47% and 100%, respectively, at a salt concentration of 1 mM. Applied at 5 mM, aluminium sulphate also provided 28% and 100% inhibition of dry rot and cavity spot, respectively. Aluminium chloride (5 mM) reduced dry rot by 25% whereas aluminium lactate (5 mM) decreased cavity spot lesions by 86%. These results indicate that various aluminium‐containing salts may provide an alternative to the use of synthetic fungicides to control these pathogens.     

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice

Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion‐limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM‐IBP fusion polypeptides composed of a carbohydrate‐binding module family 11 (CBM11) and an iron‐binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM‐IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM‐IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM‐IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.     

Proteomic profiling of L-cysteine induced selenite resistance in Enterobacter sp. YSU

Background  

Enterobacter sp. YSU is resistant to several different heavy metal salts, including selenite. A previous study using M-9 minimal medium showed that when the selenite concentration was 100,000 times higher than the sulfate concentration, selenite entered Escherichia coli cells using two pathways: a specific and a non-specific pathway. In the specific pathway, selenite entered the cells through a yet to be characterized channel dedicated for selenite. In the non-specific pathway, selenite entered the cells through a sulfate permease channel. Addition of L-cystine, an L-cysteine dimer, appeared to indirectly decrease selenite import into the cell through the non-specific pathway. However, it did not affect the level of selenite transport into the cell through the specific pathway.