SMOOTH TEMPERATURE DECREASING FOR NITROGEN REMOVAL IN COLD (9-15° C) ANAMMOX BIOFILM REACTOR TESTS

For N-rich wastewater treatment the anaerobic ammonium oxidation (anammox) and nitritation-anammox (deammonification) processes are widely used. In a deammonification moving bed biofilm reactor (MBBR) a maximum total nitrogen removal rate (TNRR) of 1.5 g N m-2d-1(0.6 kg N m-3d-1) was achieved. During biofilm cultivation, temperature was gradually lowered by 0.5° C per week, and a similar TNRR was sustained at 15° C. qPCR analysis showed an increase in Candidatus Brocadia quantities from 5×103 to 1×107 anammox gene copies g-1 TSS despite temperature lowered to 15° C. Fluctuations in TNRR were rather related to changes in influent NH4+ concentration. To study the short-term effect of temperature on the TNRR, a series of batch-scale experiments were performed which showed sufficient TNRRs even at 9-15° C (4.3-5.4 mg N L-1 h-1, respectively) with anammox temperature constants ranging 1.3-1.6. After biomass was adapted to 15° C, the decrease in TNRR in batch tests at 9° C was lower (15-20%) than for biomass adapted to 17-18° C. Our experiments show that a biofilm of a deammonification reactor adapted to 15° C successfully tolerates shortterm cold shocks down to 9° C retaining a high TNRR

Increased Biomass Yield of <em>Lactococcus lactis</em> by Reduced Overconsumption of Amino Acids and Increased Catalytic Activities of Enzymes

<div><p>Steady state cultivation and multidimensional data analysis (metabolic fluxes, absolute proteome, and transcriptome) are used to identify parameters that control the increase in biomass yield of <em>Lactococcus lactis</em> from 0.10 to 0.12 C-mol C-mol⁻¹ with an increase in specific growth rate by 5 times from 0.1 to 0.5 h⁻¹. Reorganization of amino acid consumption was expressed by the inactivation of the arginine deiminase pathway at a specific growth rate of 0.35 h⁻¹ followed by reduced over-consumption of pyruvate directed amino acids (asparagine, serine, threonine, alanine and cysteine) until almost all consumed amino acids were used only for protein synthesis at maximal specific growth rate. This balanced growth was characterized by a high glycolytic flux carrying up to 87% of the carbon flow and only amino acids that relate to nucleotide synthesis (glutamine, serine and asparagine) were consumed in higher amounts than required for cellular protein synthesis. Changes in the proteome were minor (mainly increase in the translation apparatus). Instead, the apparent catalytic activities of enzymes and ribosomes increased by 3.5 times (0.1 vs 0.5 h⁻¹). The apparent catalytic activities of glycolytic enzymes and ribosomal proteins were seen to follow this regulation pattern while those of enzymes involved in nucleotide metabolism increased more than the specific growth rate (over 5.5 times). Nucleotide synthesis formed the most abundant biomonomer synthetic pathway in the cells with an expenditure of 6% from the total ATP required for biosynthesis. Due to the increase in apparent catalytic activity, ribosome translation was more efficient at higher growth rates as evidenced by a decrease of protein to mRNA ratios. All these effects resulted in a 30% decrease of calculated ATP spilling (0.1 vs 0.5 h⁻¹). Our results show that bioprocesses can be made more efficient (using a balanced metabolism) by varying the growth conditions.</p> </div

Simplified scheme of carbon flux rates.

<p>Fluxes are shown in C-mmol (gdw*h)⁻¹ from A-stat experiments of <i>Lactococcus lactis</i>. Blue line represents average values of three independent experiments and red lines represent upper and lower values of standard deviations. Originally input values were experimentally measured at 20 time points and the other points were extrapolated between the measured values to calculate metabolic fluxes at interval of 0.01 h⁻¹. Violet boxes are substrates, orange boxes are products and blue boxes intracellular metabolites. Diamonds illustrates proteins and pathways involved in the given conversion of metabolites. ace - acetate, lact - lactate, etOH - ethanol, Glc - glucose, Orn - ornithine, Glx - glutamate + glutamine, Asx - aspartate + asparagine, PPP - peptose phosphate pathway, Pyr - pyrimidine synthesis, Pur - purine synthesis, I_AA_Glu_Group - the sum of consumption of arginine, proline, glutamine and glutamate, I_AA_Ala_Group - the sum of consumption of alanine, asparagine, aspartate, cysteine, glycine, serine and threonine, I_AA_His_Group - the sum of consumption of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, tyrosine and valine, X_prod_Pyr - unmeasured products to balance carbon in the calculations, X_prod_AA3 - unmeasured products from histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, tyrosine and valine to balance carbon in the calculations and X_prod_Glu - unmeasured products from arginine, proline, glutamine and glutamate to balance carbon in the calculations.</p

Changes of protein abundances and cost of ATP.

<p>Abundances are present in copies fl⁻¹ and cost of ATP in ATP fl⁻¹. Ten the most abundant pathways/cellular processes with increase of specific growth rate are illustrated. Distribution has been made according to the classification by Bolotin <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048223#pone.0048223-Bolotin1" target="_blank">[23]</a>. All data and distribution according to BioCyc database can be seen in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048223#pone.0048223.s002" target="_blank">File S2</a>, Tables S9 and S10.</p

Distribution of proteins.

<p>All proteins quantified at all specific growth rates has been divided into different groups by function. Proteins in the network covers enzymes that are involved in the reactions used for metabolic flux analysis and listed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048223#pone.0048223.s001" target="_blank">File S1</a>, Table S1. Lists of all proteins can be seen in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048223#pone.0048223.s002" target="_blank">File S2</a>, Table S8.</p

Distribution of protein concentrations.

<p>Concentrations (copies fl⁻¹) of proteins at specific growth rate 0.2 h⁻¹ are shown. Lower picture shows top 40 proteins fl-1 at the same specific growth rate.</p

Self-reproduction and doubling time limits of different cellular subsystems

Abstract Ribosomes which can self-replicate themselves practically autonomously in beneficial physicochemical conditions have been recognized as the central organelles of cellular self-reproduction processes. The challenge of cell design is to understand and describe the rates and mechanisms of self-reproduction processes of cells as of coordinated functioning of ribosomes and the enzymatic networks of different functional complexity that support those ribosomes. We show that doubling times of proto-cells (ranging from simplest replicators up to those reaching the size of E. coli) increase rather with the number of different cell component species than with the total numbers of cell components. However, certain differences were observed between cell components in increasing the doubling times depending on the types of relationships between those cell components and ribosomes. Theoretical limits of doubling times of the self-reproducing proto-cells determined by the molecular parameters of cell components and cell processes were in the range between 6–40 min