Chitin: a potential new alternative nitrogen source for the tertiary,algal-based treatment of pulp and paper mill wastewater

Every day, pulp and paper mills in the USA discharge millions of liters of wastewater. Primary and secondary treatment of this wastewater often enriches it with phosphorus, resulting in uncontrolled eutrophication of receiving water bodies. A new method of tertiary wastewater treatment uses controlled growth of algae in a photobioreactor to sequester phosphorus into algal biomass, which is then harvested. This typically requires addition of a nitrogen fertilizer (nitrate, ammonium, or urea) to the water. We show on the laboratory scale that chitin can be used as an alternative source of nitrogen for the tertiary treatment of pulp mill wastewater using algae. We demonstrate that phosphorus can be efficiently removed from pulp wastewater using algae and chitin. Furthermore, phosphorus removal with chitin did not result in an increase in dissolved nitrogen in the wastewater because it is insoluble, unlike conventional nitrogen fertilizers. Despite its insolubility, it has recently been found that many diverse algae and cyanobacteria can use it as a source of nitrogen. Chitin has many advantages over conventional nitrogen fertilizers for use in wastewater treatment technologies. It is the second-most abundant natural polymer and is a waste product of the shellfish industry. Chitin is sustainable, inexpensive, and carbon neutral. Thus, chitin improves the sustainability and carbon footprints associated with water treatment, while the production of commercially attractive algal biomass helps to offset costs associated with the water treatment system itself.     

Proteolytic cleavage of encephalomyocarditis virus capsid region substrates by precursors to the 3C enzyme.

Picornavirus protease 3C is normally released from its P3 precursor by two successive self-cleavage reactions. The free enzyme can then catalyze most of the remaining processing events within the viral polyprotein. To investigate the role of the 3C precursors in the processing cascade, we constructed cDNA clones which expressed genetically altered forms of the encephalomyocarditis P3 region in vitro. Site-specific substitutions were introduced into the Gln-Gly residues at the 3B-3C and 3C-3D junctions, and the resulting proteins were tested for their ability to self-process and to catalyze cleavage of viral capsid precursors in cell-free protease assays. We determined that three P3 region precursor proteins (3ABC, 3CD, and P3), harboring inactive cleavage sites, were as active as the free enzyme (3C) in processing assays with capsid substrates. Further, we found that in addition to the naturally occurring Gln-Gly and Gln-Ser amino acid pairs, the encephalomyocarditis 3C enzyme was able to process Gln-Cys but not Gln-Thr, Gln-Ile, Gln-Tyr, Arg-Gly, or Leu-Gly combinations when these residues were substituted into normal cleavage site contexts.     

Effect of Altered Sterol Composition on Growth Characteristics of Saccharomyces cerevisiae

The function of sterols in mitochondrial structures of yeast was examined. Sterol mutant strains were employed to examine the effects of altered sterolic content on optimal and permissive growth temperatures in respiring and fermenting cultures. Although fermentative growth was unaffected by sterol composition, a definite decrease in both the optimal and the permissive growth temperatures of respiring cultures was observed when ergosterol was replaced by Delta(8(9), 22)-ergostadiene-3beta-ol. In vitro studies showed a similar decrease in membrane phase transition temperatures of the mitochondrial enzyme S-adenosylmethionine: Delta(24)-sterol methyltransferase in the mutant strains. Increased sterol and methyltransferase levels were detected in strains incapable of synthesizing ergosterol. A possible control function governing sterol synthesis is proposed for ergosterol.     

Cell Division and Deoxyribonucleic Acid Synthesis after a Nutritional Shift-Up of Saccharomyces cerevisiae

Rates of cell division and deoxyribonucleic acid synthesis after shift-up with grande and mitchondrial deoxyribonucleic-acid-less petite yeasts were studied. The results indicate that simple eukaryotes behave as prokaryotes.     

Transmethylation of Sterols in Aerobically Adapting Saccharomyces cerevisiae

The transmethylation of methyl-(14)C-methionine and methyl-(14)C-adenosylmethionine into the nonsaponifiable lipids of anaerobically grown yeast during adaptation to aerobic conditions was investigated. The rate and extent of methyl transfer increased with aeration time and was dependent upon the presence of a fermentable carbon source and O(2). Methionine and adenosylmethionine uptake rates increased in adaptation buffer but did not seem to be the rate-limiting factor for transmethylation under the conditions studied. Thinlayer chromatography of the nonsaponifiable fraction after exposure to label showed the labeled product to be ergosterol. Samples taken after short-term exposure to label were composed of two labeled steroidal products, one with kinetics of an ergosterol precursor.     

Serine transhydroxymethylase in methionine biosynthesis in Saccharomyces cerevisiae

Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the beta-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme.     

Differential Effect of Respiratory Inhibitors on Ergosterol Synthesis by Saccharomyces cerevisiae During Adaptation to Oxygen

The effect of different respiratory inhibitors on the ergosterol content of microaerobically grown non-proliferating yeast cultures was monitored during adaptation to oxygen. It was found that dinitrophenol, azide, and cyanide, which act on the mechanism of the respiratory chain, cause a marked stimulation of sterol production. Acriflavine and chloramphenicol, which affect the synthesis of the respiratory apparatus, caused a delay in the onset of ergosterol synthesis or a marked decrease in sterol content. The data obtained provide presumptive evidence that a component of sterol formation is synthesized on the 70S ribosomal system of the mitochondrion and induced in the presence of oxygen.     

Modulation of the induction and circumvention of immunological tolerance to human gamma-globulin by interleukin 1

As with agents capable of causing the release of IL 1, IL 1 itself is capable of modulating certain tolerance-inducing events. Under the condition used in the present study, it previously has been firmly established that injection of A/J mice with DHGG induces a state of antigen-specific tolerance in both T helper (Th) and B cells. The tolerance in the B cell is of long duration, whereas that in the B cell is of shorter duration. Recombinant IL 1 (rIL 1) given shortly after the tolerogen DHGG results in the inhibition of the induction of tolerance resulting in antibody production. The induction of tolerance is inhibited at both its antigen-specific Th cell and B cell levels, although the latter may be caused by the former. The inhibition of the induction of tolerance by rIL 1 is not correlated to the generation of antigen-specific T suppressor cells. IL 1 mimics lipopolysaccharide and 8-bromoguanosine, which generate IL 1 production, in its ability to interfere with the in vivo induction of tolerance. However, in contrast to these latter mitogens which cause both terminal differentiation of B cells and IL 1 production, IL 1 itself does not cause in vivo circumvention of long-term tolerant Th cells in the presence of competent B cells and antigen. These latter findings suggest that a signal(s) in addition to those delivered by IL 1 is required for activation of the B cell compartment recovering from tolerance to antibody production. AHGG (immunogen) is a potent generator of IL 1 release, whereas DHGG has no effect on IL 1 release from macrophages and AHGG inhibits the induction of tolerance by DHGG. These latter results suggest that the lack of an IL 1 signal may be responsible for the deliverance of a tolerogenic rather than an immunogenic signal to the Th cell.