Potential of municipal solid waste paper as raw material for production of cellulose nanofibres.

When aiming for higher resource efficiency, greater utilization of waste streams is needed. In this work, waste paper separated from mixed municipal solid waste (MSW) was studied as a potential starting material for the production of cellulose nanofibres (CNFs). The waste paper was treated using three different techniques, namely pulping, flotation and washing, after which it was subjected to an ultrafine grinding process to produce CNFs. The energy consumption of the nanofibrillation and nanofibre morphology, as well as properties of the prepared nanofibers, were analysed. Despite the varying amounts of impurities in the waste fibres, all samples could be fibrillated into nanoscale fibres. The tensile strengths of the CNF networks ranged from 70 to 100 MPa, while the stiffness was ∼7 GPa; thus, their mechanical strength can be adequate for applications in which high purity is not required. The contact angles of the CNF networks varied depending on the used treatment method: the flotation-treated networks were more hydrophilic (contact angle 52.5°) and the washed networks were more hydrophobic (contact angle 72.6°).

Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110.

BACKGROUND: Norvaline is an unusual non-proteinogenic branched-chain amino acid which has been of interest especially during the early enzymological studies on regulatory mutants of the branched-chain amino acid pathway in Serratia marcescens. Only recently norvaline and other modified amino acids of the branched-chain amino acid synthesis pathway got attention again when they were found to be incorporated in minor amounts in heterologous proteins with a high leucine or methionine content. Earlier experiments have convincingly shown that norvaline and norleucine are formed from pyruvate being an alternative substrate of alpha-isopropylmalate synthase, however so far norvaline accumulation was not shown to occur in non-recombinant strains of E. coli. RESULTS: Here we show that oxygen limitation causes norvaline accumulation in E. coli K-12 W3110 during grow in glucose-based mineral salt medium. Norvaline accumulates immediately after a shift to oxygen limitation at high glucose concentration. On the contrary free norvaline is not accumulated in E. coli W3110 in aerobic cultures. The analysis of medium components, supported by transcriptomic studies proposes a purely metabolic overflow mechanism from pyruvate into the branched chain amino acid synthesis pathway, which is further supported by the significant accumulation of pyruvate after the oxygen downshift. The results indicate overflow metabolism from pyruvate as necessary and sufficient, but deregulation of the branched chain amino acid pathway may be an additional modulating parameter. CONCLUSION: Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over alpha-ketobutyrate and alpha-ketovalerate.Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the physiology of E. coli under oxygen limitation in general.

High cell density media for Escherichia coli are generally designed for aerobic cultivations - consequences for large-scale bioprocesses and shake flask cultures.

BACKGROUND: For the cultivation of Escherichia coli in bioreactors trace element solutions are generally designed for optimal growth under aerobic conditions. They do normally not contain selenium and nickel. Molybdenum is only contained in few of them. These elements are part of the formate hydrogen lyase (FHL) complex which is induced under anaerobic conditions. As it is generally known that oxygen limitation appears in shake flask cultures and locally in large-scale bioreactors, function of the FHL complex may influence the process behaviour. Formate has been described to accumulate in large-scale cultures and may have toxic effects on E. coli.Although the anaerobic metabolism of E. coli is well studied, reference data which estimate the impact of the FHL complex on bioprocesses of E. coli with oxygen limitation have so far not been published, but are important for a better process understanding. RESULTS: Two sets of fed-batch cultures with conditions triggering oxygen limitation and formate accumulation were performed. Permanent oxygen limitation which is typical for shake flask cultures was caused in a bioreactor by reduction of the agitation rate. Transient oxygen limitation, which has been described to eventually occur in the feed-zone of large-scale bioreactors, was mimicked in a two-compartment scale-down bioreactor consisting of a stirred tank reactor and a plug flow reactor (PFR) with continuous glucose feeding into the PFR.In both models formate accumulated up to about 20 mM in the culture medium without addition of selenium, molybdenum and nickel. By addition of these trace elements the formate accumulation decreased below the level observed in well-mixed laboratory-scale cultures. Interestingly, addition of the extra trace elements caused accumulation of large amounts of lactate and reduced biomass yield in the simulator with permanent oxygen limitation, but not in the scale-down two-compartment bioreactor. CONCLUSION: The accumulation of formate in oxygen limited cultivations of E. coli can be fully prevented by addition of the trace elements selenium, nickel and molybdenum, necessary for the function of FHL complex. For large-scale cultivations, if glucose gradients are likely, the results from the two-compartment scale-down bioreactor indicate that the addition of the extra trace elements is beneficial. No negative effects on the biomass yield or on any other bioprocess parameters could be observed in cultures with the extra trace elements if the cells were repeatedly exposed to transient oxygen limitation.

Fermentation process for tetrameric human collagen prolyl 4-hydroxylase in Escherichia coli: improvement by gene optimisation of the PDI/beta subunit and repeated addition of the inducer anhydrotetracycline.

The collagen prolyl 4-hydroxylases (C-P4Hs) that reside within the lumen of the endoplasmic reticulum (ER) are the key enzymes in the biosynthesis of collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers consisting of two catalytic alpha subunits and two beta subunits that are identical to protein disulfide isomerase (PDI). Cytoplasmic production of an active human C-P4H has recently been described in the Origami (trxB gor) mutant Escherichia coli using a bicistronic vector with independent control of the alpha and PDI/beta subunit expression by the tetA and T5-lac promoters, respectively, enabling sequential induction (Neubauer, A., Neubauer, P., Myllyharju, J., 2005. High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli. Matrix Biol. 24, 59-68). We show here that the yield of active C-P4H in shake flasks is increased 50-fold by improving the expression level of the PDI/beta subunit through gene optimisation. We also found that stable expression of the alpha subunit mRNA in a fed-batch fermentation process requires repeated additions of anhydrotetracycline. This finding may be of a wider general importance for the use of the tetA promoter in fed-batch cultivations, especially if recombinant proteins are expressed during long production phases. We also show that growth of the E. coli Origami strain to high cell density on a complex medium with consecutive sequential induction is difficult to achieve and that optimisation of similarly complicated systems can greatly benefit from the use of quantitative mRNA analysis for the evaluation of transcriptional bottlenecks. The optimisation approach resulted in a fermentation yield of 143 mg L(-1) of active C-P4H, corresponding to approximately 7.5% of the total soluble cell protein.

Transient increase of ATP as a response to temperature up-shift in Escherichia coli.

SUMMARY: BACKGROUND: Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift. RESULTS: The current study shows that a temperature up-shift is followed by a very fast significant transient increase of the cellular ATP concentration within the first minutes. This increase is connected to a longer lasting elevation of the cellular respiration and glucose uptake. Also the mRNA level of typical heat shock genes increases within only one minute after the heat-shock. CONCLUSION: The presented data prove the very fast response of E. coli to a heat-shock and that the initial response includes the increase of the ATP pool which is important to fulfil the need of the cell for new syntheses, as well as for the function of chaperones and proteases.