Andrus Seiman

Step-wise temperature decreasing cultivates a biofilm with high nitrogen removal rates at 9°C in short-term anammox biofilm tests

ABSTRACT The anaerobic ammonium oxidation (anammox) and nitritation-anammox (deammonification) processes are widely used for N-rich wastewater treatment. When deammonification applications move towards low temperature applications (mainstream wastewater has low temperature), temperature effect has to be studied. In current research, in a deammonification moving bed biofilm reactor a maximum total nitrogen removal rate (TNRR) of 1.5 g N m−2 d−1 (0.6 kg N m−3 d−1) was achieved. Temperature was gradually lowered by 0.5°C per week, and a similar TNRR was sustained at 15°C during biofilm cultivation. Statistical analysis confirmed that a temperature decrease from 20°C down to 15° did not cause instabilities. Instead, TNRR rose and treatment efficiency remained stable at lower temperatures as well. Quantitative polymerase chain reaction analyses showed an increase in Candidatus Brocadia quantities from 5 × 103 to 1 × 107 anammox gene copies g−1 total suspended solids (TSS) despite temperature lowered to 15°C. Fluctuations in TNRR were rather related to changes in influent 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 (1.24–3.43 mg N g−1 TSS h−1, respectively) with anammox temperature constants (Q10) ranging 1.3–1.6. Experiments showed that a biofilm adapted to 15°C can perform N-removal most sufficiently at temperatures down to 9°C as compared with biofilm adapted to higher temperature. After biomass was adapted to 15°C, the decrease in TNRR in batch tests at 9°C was lower (15–20%) than that for biomass adapted to 17–18°C.

A portable capillary electropherograph equipped with a cross-sampler and a contactless-conductivity detector for the detection of the degradation products of chemical warfare agents in soil extracts

A fully portable CE device equipped with a capacitively coupled contactless‐conductivity detector and a cross‐injection device is put to the test in laboratory conditions. The portable device is capable of working on batteries for at least 4 h. After that, its performance is strongly affected by the drop in the high‐voltage output and analysis may be interrupted if its length exceeds a reasonable time. The concentration of the BGE affects both ionic strength and conductivity. Choosing an optimal concentration of BGE is therefore about finding a good compromise between selectivity and sensitivity. All experiments were performed using a mixture of histidine and MES with a concentration of 15 mM as BGE. The performance of the cross‐injection device is optimized by the use of internal standards. Satisfactory reproducibility is gained as the RSD of peak areas is reduced to 8% or less. LODs for different phosphonic acids are in the range of 2.5–9.7 μM. For the analysis of adsorption of phosphonic acids in sand and loamy soil samples, calibration curves are constructed. Linearity in a measured concentration range of 10–100 μM is excellent, as the squares of correlation constants are ∼1. The concentration analysis of phosphonic acids in soil extracts demonstrates that their adsorption curves in sand and loamy soil follow different adsorption isotherms.

Monitoring of the electroosmotic flow of ionic liquid solutions in non-aqueous media using thermal marks

The possibility of applying a new method employing thermal marks to measuring the rate of the electroosmotic flow (EOF) in non-aqueous capillary electrophoresis (NACE) was investigated. The thermal marks were monitored by using a contactless conductivity detection. During one experiment and in between the series of experiments the reproducibility of the method was excellent. The EOF rate was measured 4-7 times during one experiment, the precision of measurement being around 0.5%. In this study, the influence of 1-butyl-3-methyl-imidazolium salts in organic solvents on the rate of the EOF was investigated. Various organic solvents were mixed with an ionic liquid of various concentrations and the EOF rate was measured using thermal marks. The accuracy of the method was compared with that of the neutral marker one. Five benzoic acid derivatives were separated while the EOF was monitored. The relative standard deviations of the corrected effective mobilities of the above analytes were in the range of 1.0-6.1%.

Nitritating-anammox biomass tolerant to high dissolved oxygen concentration and C/N ratio in treatment of yeast factory wastewater

Maintaining stability of low concentration (<1 g L−1) floccular biomass in the nitritation-anaerobic ammonium oxidation (anammox) process in the sequencing batch reactor (SBR) system for the treatment of high COD (>15,000 mg O2 L−1) to N (1680 mg N L−1) ratio real wastewater streams coming from the food industry is challenging. The anammox process was suitable for the treatment of yeast factory wastewater containing relatively high and abruptly increased organic C/N ratio and dissolved oxygen (DO) concentrations. Maximum specific total inorganic nitrogen (TIN) loading and removal rates applied were 600 and 280 mg N g−1 VSS d−1, respectively. Average TIN removal efficiency over the operation period of 270 days was 70%. Prior to simultaneous reduction of high organics (total organic carbon>600 mg L−1) and N concentrations>400 mg L−1, hydraulic retention time of 15 h and DO concentrations of 3.18 (±1.73) mg O2 L−1 were applied. Surprisingly, higher DO concentrations did not inhibit the anammox process efficiency demonstrating a wider application of cultivated anammox biomass. The SBR was fed rapidly over 5% of the cycle time at 50% volumetric exchange ratio. It maintained high free ammonia concentration, suppressing growth of nitrite-oxidizing bacteria. Partial least squares and response surface modelling revealed two periods of SBR operation and the SBR performances change at different periods with different total nitrogen (TN) loadings. Anammox activity tests showed yeast factory-specific organic N compound-betaine and inorganic N simultaneous biodegradation. Among other microorganisms determined by pyrosequencing, anammox microorganism (uncultured Planctomycetales bacterium clone P4) was determined by polymerase chain reaction also after applying high TN loading rates.

Start-up of low-temperature anammox in UASB from mesophilic yeast factory anaerobic tank inoculum

Robust start-up of the anaerobic ammonium oxidation (anammox) process from non-anammox-specific seeding material was achieved by using an inoculation with sludge-treating industrial -, organics- and N-rich yeast factory wastewater. N-rich reject water was treated at 20°C, which is significantly lower than optimum treatment temperature. Increasing the frequency of biomass fluidization (from 1–2 times per day to 4–5 times per day) through feeding the reactor with higher flow rate resulted in an improved total nitrogen removal rate (from 100 to 500 g m−3d−1) and increased anammox bacteria activity. As a result of polymerase chain reaction (PCR) tests, uncultured planctomycetes clone 07260064(4)-2-M13-_A01 (GenBank: JX852965) was identified from the biomass taken from the reactor. The presence of anammox bacteria after cultivation in the reactor was confirmed by quantitative PCR (qPCR); an increase in quantity up to ∼2×106 copies g VSS−1 during operation could be seen in qPCR. Statistical modelling of chemical parameters revealed the roles of several optimized parameters needed for a stable process.

Improving precision of manual hydrodynamic injection in capillary electrophoresis with contactless conductivity detection.

Reproducible injection in capillary electrophoresis has been difficult to achieve with manual injection techniques using simple injection devices, such as gravity injection (siphoning) or hydrodynamic sample splitting. We demonstrate that the injection reproducibility can be improved using very simple means. With hydrodynamic sample splitter, a passive micro-metering valve can be inserted in-line to regulate the sample flow rate through the splitter interface. A significant improvement of both reproducibility and repeatability was achieved. The reproducibility of RSD of the peak areas improved from 25.4% to 4.4%, while the repeatability was below 4.1% when micro-metering valve was used. Additional simple correction that can be used to further improve the variability of injected sample volumes in any hydrodynamic injection mode in CE with conductivity detection was proposed and verified. The measured EOF peak can serve as a simple indicator of the injected volume and can be effectively used for additional correction. By a linear function between the injection volume and the peak area of the EOF, the RSD values of peak areas for both manual gravity injection and hydrodynamic sample splitter were further improved below 2% RSD. The linearity of the calibration curve was also significantly improved. The proposed correction works even with slight differences in matrix composition, as demonstrated on the analysis aqueous soil extract of model mixture of five nerve agent degradation products.

Protein turnover forms one of the highest maintenance costs in Lactococcus lactis.

Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (μ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at μ = 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for ~75% of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35%. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47% of the total energy produced at μ = 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.

Nutritional requirements and media development for Lactococcus lactis IL1403

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.

Increased biomass yield of Lactococcus lactis by reduced overconsumption of amino acids and increased catalytic activities of enzymes.

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 Lactococcus lactis 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.

The detection of nerve agent degradation products in different soil fractions using capillary electrophoresis with contactless conductivity detection

The adsorption of various phosphonic acids in sand and loam was studied. Samples of both soil types were sieved into three different fractions according to particle size. The fractions used were in the range 0–100, 100–200 and 200–400 μm. The performance of the capillary electrophoresis equipped with contactless conductivity detection was investigated. The limit of detection for the phosphonic acids tested was in the range 0.11–1.4 ppm. Different isotherms were constructed for all adsorption curves. Adsorption was found to be higher in sand than in loam when the Langmuir adsorption isotherm was used. The adsorption of methylphosphonic acid was higher than that of other phosphonic acids due to the smaller molecular size of the former.