Accumulation and degradation of polyphosphate in Acinetobacter sp.

Biological phosphate removal from waste water is a biotechnological alternative to chemical phosphorus precipitation. This process is obtained by recycling the sludge through anaerobic and aerobic zones. In the anaerobic parts phosphate is released by the sludge and during anaerobiosis phosphate is taken up. Biological phosphate removal is dependent on the enrichment of activated sludge with polyphosphate accumulating Acinetobacter . Like activated sludge, pure cultures of strictly aerobic Acinetobacter sp. absorbe phosphate (up to 100 mg phosphorus per g dry biomass) during aerobic conditions and release it anaerobically. The aim of this study was to gather knowledge on the uptake and release of phosphate by Acinetobacter and the metabolic functions of polyphosphate.The accumulation of polyphosphate by pure cultures of Acinetobacter strain 210A depended on the presence of an intra- or extracellular energy source (chapter 2). The highest amount of polyphosphate was found in cells in which energy supply was not limited, namely at low growth rates under sulphur limitation, and in the stationary phase of growth when either the nitrogen or the sulphur source was depleted. Accumulation of polyphosphate was also possible during endogenous respiration. When this respiration was blocked with KCN the phosphate uptake stopped, while the inhibition of the protein synthesis with streptomycin enhanced the accumulation of phosphate, which indicated the competition between protein synthesis and polyphosphate synthesis for energy. There was a pronounced effect of the temperature on phosphorus accumulation but this effect varied from strain to strain.The role and behaviour of cations in the accumulation and release of phosphate was studied (chapter 3). PO 43-was released together with 1.8 protons. Mg 2+appeared to be the most important counterion of polyphosphate in Acinetobacter strain 210A. It was released and taken up simultaneously with phosphate. Mg 2+was not an essential polyphosphate counterion. If Mg 2+was depleted, stationary cultures of Acinetobacter strain 210A took up the same amount of phosphate with Ca 2+as the most important counterion. In the presence of Mg 2+stationary cultures did not need Ca 2+for their phosphate absorption, but the presence of K +seemed to be crucial for this process, although this cation did not play a quantitatively important role as a polyphosphate counterion. In addition, the influx and efflux of K +was independent of phosphate uptake and release. Continuous cultivation at low growth rates under K +-limitation did not result in polyphosphate accumulation, while under substrate or Mg 2+- limitation large amounts of polyphosphate were present in the cells. The same effect was found in activated sludge. 5 mg K +per litre was needed for a satisfactory biological phosphate removal in the aerobic zone of a wastewater treatment plant. Granules of Mgpolyphosphate in Acinetobacter strain 210A could serve as a Mg 2+-reserve. Cells with these granules were able to grow in a medium free of Mg 2+, whereas cells without granules were not, they only grew in the presence of extracellular Mg 2+. Polyphosphate in cell-free extracts of Acinetobacter strain 210A could be degraded by the enzymes polyphosphatase or polyphosphate:AMP phosphotransferase (chapters 4 and 6). Polyphosphate glucokinase, polyphosphate dependent NAD-kinase and polyphosphatekinase were not detectable. Polyphosphate:AMP phosphotransferase was also found in Acinetobacter strain B8, but not in Acinetobacter strain P, which contained only polyphosphatekinase. Both strains were able to accumulate large amounts of polyphosphate. In strains that cannot accumulate this biopolymer, no or very small activities of polyphosphatekinase and polyphosphate: AMP phosphotransferase were found. All strains showed activities of adenylate kinase. It was demonstrated that by the combined action of polyphosphate:AMP phosphotransferase and adenylate kinase a continuous regeneration of ATP from AMP or ADP was possible as long as polyphosphate was present. Polyphosphate:AMP phosphotransferase could use native and synthetic polyphosphate as substrate and showed a maximum activity at a pH of 8.5. Its activity was stimulated by (NH 4 ) 2 SO 4 , the K m for AMP appeared to be 0.6 mM, and V max was 60 nmol.min -1.mg -1protein. Polyphosphatase in cell-free extracts of strain 210A was able to hydrolyse native polyphosphate and synthetic Mg-polyphosphate. The K- and Na-form, however, were not degraded. The activities of polyphosphate:AMP phosphotransferase and adenylate kinase in activated sludge correlated well with the ability of the sludge to remove phosphate biologically from waste water.Degradation of polyphosphate invivo in Acinetobacter strain 210A occurred if the energy supply in the cell was stopped, for example under anaerobiosis or in the presence of KCN, α-dinitrophenol or N-N'-dicyclohexylcarbodiimid (chapter 5). The degradation and synthesis of polyphosphate was dependent on the ATP concentration in the cells. Lower ATP concentrations caused a faster phosphate release. This release was stimulated by alcohols. The transmembrane protongradient seemed to play an important role in the anaerobic energy metabolism of this strictly aerobic bacterium. Addition of α-dinitrophenol, a protonionophore, decreased the cellular ATP concentration and stimulated the polyphosphate degradation. The role of polyphosphate as an energy reserve invivo has been demonstrated by experiments in which five strains were incubated anaerobically. Cells of Acinetobacter strains 210A and B8, which were able to accumulate polyphosphate, released large amounts of ortho-phosphate anaerobically and contained high levels of ATP. Cells of two other strains of Acinetobacter and one strain of Pseudomonas which didn't accumulate polyphosphate, showed a much smaller release of phosphate and contained only low ATP concentrations. Cells of strain 210A cultivated under phosphorus limitation or at 350C did not contain detectable amounts of polyphosphate. As a result their ATP level was low and they released only small or negligible amounts of phosphate under anaerobic conditions.Mg-polyphosphate in Acinetobacter sp. is a multifunctional compound. It can serve as: (1) an energy reserve if it is degraded by a reaction with AMP, catalyzed by polyphosphate: AMP phosphotransferase, (2) a phosphorus reserve if it is hydrolyzed by polyphosphatase, and (3) a Mg 2+reserve whereby Mg 2+can be replaced by Ca 2+as a counterion. The most important role of polyphosphate in wastewater treatment plants with biological phosphate removal, and probably also in natural environments, is its use as an energy reserve to sustain temporary anaerobiosis. This property might explain the enrichment of activated sludge subjected to alternating anaerobic and aerobic conditions with polyphosphate accumulating Acinetobacter sp.

Biofuel production from acid-impregnated willow and switchgrass

As part of a broader technical and economic feasibility study, we studied production of bioethanol from two types of lignocellulosic biomass by way of concentrated acid impregnation at low temperature. Willow chips and switchgrass were submitted to various impregnation techniques with concentrated sulfuric acid at varying acid: biomass ratios and impregnation times. Goal of the experiments was to investigate the technical feasibility of concentrated acid pretreatment technology as part of an industrial process that employs recycling of acid through biological means. Experimental results showed that significant amounts of fermentable sugars including glucose (up to 78 f max. obtainable glucose) and xylose can be obtained by relatively simple impregnation techniques at room temperature. Fermentation of willow-derived hydrolysates with S. Cerevisiae yielded 0.45 - 0.49 g ethanol/g glucose. Ethanol production rates however were 38 ower compared to standard glucose fermentation, prompting the need for further optimization to reduce the formation of acetic acid and furfural, two fermentation inhibitors. Novel impregnation techniques, including employment of sulfur trioxide, were also investigated but require more work to assess technical feasibilit

Isolation and characterisation of fungi growing on volatile aromatic hydrocarbons as their sole carbon and energy source

Five fungal strains that are able to grow on toluene were isolated from enrichment cultures. Three different techniques were used: solid state-like batches, air biofilters and liquid cultures. Fungal growth in the latter systems was favoured by combining low pH and low water activity. Soil and groundwater samples from gasoline-polluted environments were used as inocula. The isolates were identified as deuteromycetes belonging to the genera Cladophialophora, Exophiala and Leptodontium and the ascomycete Pseudeurotium zonatum. The previously isolated toluene-degrading fungus Cladosporium sphaerospermum was included in the present study. Results showed that fungi grew on toluene with doubling times of about 2 to 3 days. Some of the strains also grew on ethylbenzene and styrene. The apparent half-saturation constant (Km) for toluene oxidation ranged from 5 to 22 μM. Degradation activity was inhibited by 50t toluene concentrations ranging from 2.4 to 4.7 mM. These kinetic parameters are comparable to analogous data reported for toluene-degrading bacteria. The ability of fungi to grow at low water activities and low pH suggest that they may be used for the purification of gas streams containing aromatic hydrocarbons in air biofilters

Biological off-gas treatment: let's make things better

Biological off-gas treatment is the most effective cleaning method for many off-gases which contain low concentration of pollutants (<5 g/m3). The world market share in off-gas treatment is a few percent. Potential buyers are reserved because of existing biofilter quality differences and lack of experience with biofilters. Besides that, not all pollutants can be eliminated by the use of biological methods. Nevertheless, there is much room for a growing biofilter market, in particularly because many countries are introducing clean air acts within a few years. Successful market penetration depends on the capability of companies to supply reliable biofilters and the field of operation of biofilters. Therefore, R&D is carried out world wide to improve biofilters, bioscrubbers, bio-trickling filters and membrane bioreactors

Non-chemically modified food starches

A process for producing thermally inhibited starch, specifically thermally inhibited non- pregelatinized granular starch, is described, resulting in a viscostable starch product. The process comprising providing an alkaline starch, specifically an alkaline non-pregelatinized granular starch,having a pH of at least 8; subjecting the starch to a hydrothermal treatment, specifically to obtain a hydrothermally treated non-pregelatinized granular starch, said hydrothermal treatment being at a temperature of 45 -200 °C with steam at a steam pressure of 0.1-15 bar or a gas mixture comprising water vapor at a partial water vapor pressure of 0.1-1 bar; dehydrating the starch, specifically the hydrothermally treated non-pregelatinized granular starch,to a moisture content of 2 wt% or lower and subjecting the starch to a thermal treatment by heating the starch to a temperature of 120 -190 °C to obtain viscostability,cooling and optionally further processing the starch

Non-chemically modified food starches

A process for producing thermally inhibited starch, specifically thermally inhibited non- pregelatinized granular starch, is described, resulting in a viscostable starch product. The process comprising providing an alkaline starch, specifically an alkaline non-pregelatinized granular starch,having a pH of at least 8; subjecting the starch to a hydrothermal treatment, specifically to obtain a hydrothermally treated non-pregelatinized granular starch, said hydrothermal treatment being at a temperature of 45 -200 °C with steam at a steam pressure of 0.1-15 bar or a gas mixture comprising water vapor at a partial water vapor pressure of 0.1-1 bar; dehydrating the starch, specifically the hydrothermally treated non-pregelatinized granular starch,to a moisture content of 2 wt% or lower and subjecting the starch to a thermal treatment by heating the starch to a temperature of 120 -190 °C to obtain viscostability,cooling and optionally further processing the starch