Microbial interactions shape cheese flavour formation.

Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α-ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus, highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile.

Use of Cell Envelope Targeting Antibiotics and Antimicrobial Agents as a Powerful Tool to Select for Lactic Acid Bacteria Strains With Improved Texturizing Ability in Milk Fermentations.

Many antibiotics and antimicrobial agents have the bacterial cell envelope as their primary target, interfering with functions such as synthesis of peptidoglycan, membrane stability and permeability, and attachment of surface components. The cell envelope is the outermost barrier of the bacterial cell, conferring protection against environmental stresses, and maintaining structural integrity and stability of the growing cell, while still allowing for required metabolism. In this work, inhibitory concentrations of several different cell envelope targeting antibiotics and antimicrobial agents were used to select for derivatives of lactic acid bacteria (LAB) with improved properties for dairy applications. Interestingly, we observed that for several LAB species a fraction of the isolates had improved milk texturizing capabilities. To further improve our understanding of the mechanisms underlying the improved rheology and to validate the efficacy of this method for strain improvement, genetic and physiological characterization of several improved derivatives was performed. The results showed that the identified genetic changes are diverse and affect also other cellular functions than the targeted cell surface. In short, this study describes a new versatile and powerful toolbox based on targeting of the cell envelope to select for LAB derivatives with improved phenotypic traits for dairy applications.

Going back to school - An opportunity for lifelong learning for people with dementia in Denmark (Innovative practice).

The provision of lifelong learning for older people is often promoted as a way of engaging socially and maintaining cognitive function. The concept is also used with people with dementia, but is often limited to short-term programmes. Innovative practice from Denmark takes this concept further, offering people with early stage dementia the opportunity to return to school to attend classes in cognitive training, music, art and woodcraft. A pilot study conducted by the school of teaching and communication (Voksenskolen For Undervisning og Kommunikation) offers evidence for the benefits of prolonged educational programmes for people with dementia in maintaining decision-making, cognitive function and social interactions, with limited evidence of the impact on memory. Further evidence is required to understand the impact of a person with dementia attending school as a student and to understand if this concept is transferrable to a different cultural setting.

Development of Tyrosine Decarboxylase-Negative Strains of Lactobacillus curvatus by Classical Strain Improvement.

Biogenic amines have been widely studied because of their potential toxicity in fermented foods. Several lactic acid bacteria have the potential to decarboxylate the amino acid tyrosine to tyramine. In this work, we identified two strains of Lactobacillus curvatus, Lbc1 and Lbc2, endowed with the ability to produce tyramine, a metabolic feature that limits their application in starter cultures for fermented meat. To overcome this limitation, we set out to eliminate tyramine production from L. curvatus strains by using classical strain improvement. About 4,000 mutant isolates of both strains were screened using a colorimetric method, and then potential tyrosine decarboxylase-negative mutants were selected. Firm identification of loss-of-function mutants was performed by analytical determination of tyrosine and tyramine in cultivation medium. Of the 8,000 mutants screened, only one mutant of Lbc1 and two mutants of Lbc2 had completely lost the potential to produce tyramine. Subsequently, one tyrosine decarboxylase-negative mutant of both Lbc1 and Lbc2 was characterized in more detail. DNA sequencing of the Lbc1 mutant tdcA gene disclosed two missense mutations in the promoter distal part of the coding sequence. These two mutations result in two amino acid changes in the encoded tyrosine decarboxylase, Pro87Thr and Ser130Leu, presumably inactivating the enzyme activity. The DNA sequence of the other characterized mutant, derived from strain Lbc2, showed that insertion of a 6-bp fragment at nucleotide position 1348 in the tdc gene is presumably the factor leading to loss of activity. With the successful elimination of the undesirable tyramine-producing phenotype without the use of recombinant DNA technology, these developed L. curvatus mutant strains can be safely used in the dairy industry or in the manufacture of various food products.

A framework for good biofilm reactor modeling practice (GBRMP).

A researcher or practitioner can employ a biofilm model to gain insight into what controls the performance of a biofilm process and for optimizing its performance. While a wide range of biofilm-modeling platforms is available, a good strategy is to choose the simplest model that includes sufficient components and processes to address the modeling goal. In most cases, a one-dimensional biofilm model provides the best balance, and good choices can range from hand-calculation analytical solutions, simple spreadsheets, and numerical-method platforms. What is missing today is clear guidance on how to apply a biofilm model to obtain accurate and meaningful results. Here, we present a five-step framework for good biofilm reactor modeling practice (GBRMP). The first four steps are (1) obtain information on the biofilm reactor system, (2) characterize the influent, (3) choose the plant and biofilm model, and (4) define the conversion processes. Each step demands that the model user understands the important components and processes in the system, one of the main benefits of doing biofilm modeling. The fifth step is to calibrate and validate the model: System-specific model parameters are adjusted within reasonable ranges so that model outputs match actual system performance. Calibration is not a simple 'by the numbers' process, and it requires that the modeler follows a logical hierarchy of steps. Calibration requires that the adjusted parameters remain within realistic ranges and that the calibration process be carried out in an iterative manner. Once each of steps 1 through 5 is completed satisfactorily, the calibrated model can be used for its intended purpose, such as optimizing performance, trouble-shooting poor performance, or gaining deeper understanding of what controls process performance.

Quality of care for people with multimorbidity - a case series.

BACKGROUND: Multimorbidity is becoming increasingly prevalent and presents challenges for healthcare providers and systems. Studies examining the relationship between multimorbidity and quality of care report mixed findings. The purpose of this study was to investigate quality of care for people with multimorbidity in the publicly funded healthcare system in Denmark. METHODS: To investigate the quality of care for people with multimorbidity different groups of clinicians from the hospital, general practice and the municipality reviewed records from 23 persons with multimorbidity and discussed them in three focus groups. Before each focus group, clinicians were asked to review patients' medical records and assess their care by responding to a questionnaire. Medical records from 2013 from hospitals, general practice, and health centers in the local municipality were collected and linked for the 23 patients. Further, two clinical pharmacologists reviewed the appropriateness of medications listed in patient records. RESULTS: The review of the patients' records conducted by three groups of clinicians revealed that around half of the patients received adequate care for the single condition which prompted the episode of care such as a hospitalization, a visit to an outpatient clinic or the general practitioner. Further, the care provided to approximately two-thirds of the patients did not take comorbidities into account and insufficiently addressed more diffuse symptoms or problems. The review of the medication lists revealed that the majority of the medication lists contained inappropriate medications and that there were incongruity in medication listed in the primary and secondary care sector. Several barriers for providing high quality care were identified. These included relative short consultation times in general practice and outpatient clinics, lack of care coordinators, and lack of shared IT-system proving an overview of the treatment. CONCLUSIONS: Our findings reveal quality of care deficiencies for people with multimorbidity. Suggestions for care improvement for people with multimorbidity includes formally assigned responsibility for care coordination, a change in the financial incentive structure towards a system rewarding high quality care and care focusing on prevention of disease exacerbation, as well as implementing shared medical record systems.

Enhancing the Sweetness of Yoghurt through Metabolic Remodeling of Carbohydrate Metabolism in Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

UNLABELLED: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are used in the fermentation of milk to produce yoghurt. These species normally metabolize only the glucose moiety of lactose, secreting galactose and producing lactic acid as the main metabolic end product. We used multiple serial selection steps to isolate spontaneous mutants of industrial strains of S. thermophilus and L. delbrueckii subsp. bulgaricus that secreted glucose rather than galactose when utilizing lactose as a carbon source. Sequencing revealed that the S. thermophilus strains had mutations in the galKTEM promoter, the glucokinase gene, and genes encoding elements of the glucose/mannose phosphotransferase system (PTS). These strains metabolize galactose but are unable to phosphorylate glucose internally or via the PTS. The L. delbrueckii subsp. bulgaricus mutants had mutations in genes of the glucose/mannose PTS and in the pyruvate kinase gene. These strains cannot grow on exogenous glucose but are proficient at metabolizing internal glucose released from lactose by ß-galactosidase. The resulting strains can be combined to ferment milk, producing yoghurt with no detectable lactose, moderate levels of galactose, and high levels of glucose. Since glucose tastes considerably sweeter than either lactose or galactose, the sweetness of the yoghurt is perceptibly enhanced. These strains were produced without the use of recombinant DNA technology and can be used for the industrial production of yoghurt with enhanced intrinsic sweetness and low residual levels of lactose. IMPORTANCE: Based on a good understanding of the physiology of the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, we were able, by selecting spontaneously occurring mutants, to change dramatically the metabolic products secreted into the growth medium. These mutants consume substantially more of the lactose, metabolize some of the galactose, and secrete the remaining galactose and most of the glucose back into the milk. This allows production of yoghurt with very low lactose levels and enhanced natural sweetness, because humans perceive glucose as sweeter than either lactose or galactose.

The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology.

The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 ß-d-xylosidase/α-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12.

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a ß-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-ß-d-xylopyranoside (pNPX), para-nitrophenyl-α-L-arabinofuranoside (pNPA), ß-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2-4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I - V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40°C and at pH 4.0-8.0 and showed maximum activity at pH 5.5 and 50°C. Km and kcat for pNPX were 15.6 ± 4.2 mM and 60.6 ± 10.8 s-1, respectively, and substrate inhibition became apparent above 18 mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for ß-d-xylosidase (XynB3) from Geobacillus stearothermophilus T‒6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn2+ and Ag+, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg505Tyr506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus.

Rethinking wastewater characterisation methods for activated sludge systems - a position paper.

Increasingly stringent effluent limits and an expanding scope of model system boundaries beyond activated sludge has led to new modelling objectives and consequently to new and often more detailed modelling concepts. Nearly three decades after the publication of Activated Sludge Model No1 (ASM1), the authors believe it is time to re-evaluate wastewater characterisation procedures and targets. The present position paper gives a brief overview of state-of-the-art methods and discusses newly developed measurement techniques on a conceptual level. Potential future paths are presented including on-line instrumentation, promising measuring techniques, and mathematical solutions to fractionation problems. This is accompanied by a discussion on standardisation needs to increase modelling efficiency in our industry.

Lactobacillus delbrueckii subsp. jakobsenii subsp. nov., isolated from dolo wort, an alcoholic fermented beverage in Burkina Faso.

Lactobacillus delbrueckii is divided into five subspecies based on phenotypic and genotypic differences. A novel isolate, designated ZN7a-9(T), was isolated from malted sorghum wort used for making an alcoholic beverage (dolo) in Burkina Faso. The results of 16S rRNA gene sequencing, DNA-DNA hybridization and peptidoglycan cell-wall structure type analyses indicated that it belongs to the species L. delbrueckii. The genome sequence of isolate ZN7a-9(T) was determined by Illumina-based sequencing. Multilocus sequence typing (MLST) and split-decomposition analyses were performed on seven concatenated housekeeping genes obtained from the genome sequence of strain ZN7a-9(T) together with 41 additional L. delbrueckii strains. The results of the MLST and split-decomposition analyses could not establish the exact subspecies of L. delbrueckii represented by strain ZN7a-9(T) as it clustered with L. delbrueckii strains unassigned to any of the recognized subspecies of L. delbrueckii. Strain ZN7a-9(T) additionally differed from the recognized type strains of the subspecies of L. delbrueckii with respect to its carbohydrate fermentation profile. In conclusion, the cumulative results indicate that strain ZN7a-9(T) represents a novel subspecies of L. delbrueckii closely related to Lactobacillus delbrueckii subsp. lactis and Lactobacillus delbrueckii subsp. delbrueckii for which the name Lactobacillus delbrueckii subsp. jakobsenii subsp. nov. is proposed. The type strain is ZN7a-9(T) = DSM 26046(T) = LMG 27067(T).

Draft Whole-Genome Sequence of Bacillus sonorensis Strain L12, a Source of Nonribosomal Lipopeptides.

The Bacillus sonorensis L12 draft genome sequence is approximately 4,647,754 bp in size with a G+C content of 45.2%. Over 86% of the genome contains protein-encoding genes, including several gene clusters for de novo biosynthesis of the nonribosomal lipopeptides iturin, bacitracin, and fengycin, which could mean that the strain exhibits antifungal effects.

Method to identify potential phosphorus rate-limiting conditions in post-denitrification biofilm reactors within systems designed for simultaneous low-level effluent nitrogen and phosphorus concentrations.

Water-quality standards requiring simultaneous low level effluent N and P concentrations are increasingly common in Europe and the United States of America. Moving bed biofilm reactors (MBBRs) and biologically active filters (BAFs) have been used as post-denitrification biofilm reactors in processes designed and operated for this purpose (Boltz et al., 2010a). There is a paucity of information describing systematic design and operational protocols that will minimize the potential for phosphorus rate-limited conditions as well as a lack of information describing the interaction between these post-denitrification biofilm reactors and unit processes that substantially alter phosphorus speciation (e.g., chemically enhanced clarification). In this paper, a simple mathematical model for estimating the threshold below which P becomes rate-limiting, and the model is presented and evaluated by comparing its predictions with operational data from post-denitrification MBBRs and BAFs. Ortho-phosphorus (PO(4)-P), which is the dissolved reactive component of total phosphorus, was a primary indicator of P rate-limiting conditions in the evaluated post-denitrification biofilm reactors. The threshold below which PO(4)-P becomes the rate-limiting substrate is defined: S(PO4-P):S(NOx-N) = 0.0086 g P/g N and S(PO4-P):S(M) = 0.0013 g P/g COD. Additional analyses indicate J(NOx-N)(avg) =0.48 g/m2/d when S(PO4-P):S(NOx-N) > 0.0086, and J(NOx-N)(avg) = 0.06 g/m2/d when S(PO4-P):S(NOx-N) < 0.0086. Effluent nitrate-nitrogen plus nitrite-nitrogen concentration (S(NOx-N)) from the evaluated post-denitrification biofilm reactors began to rapidly increase when S(PO4-P):S(NOx-N) was 0.01, approximately (consistent with the rate-limitation threshold of S(PO4-P):S(NOx-N) < 0.0086 predicted by the mathematical model described in this paper). Depending on the processes used at a given WWTP, optimizing chemically enhanced clarification to increase the amount of PO(4)-P that remains in the clarifiers effluent stream, dosing phosphoric acid in the MBBR or BAF influent stream, and/or optimizing secondary process EBPR may overcome phosphorus rate-limitations in the biofilm-based post-denitrification process.

Antimicrobial susceptibility of Bacillus strains isolated from primary starters for African traditional bread production and characterization of the bacitracin operon and bacitracin biosynthesis.

Bacillus spp. are widely used as feed additives and probiotics. However, there is limited information on their resistance to various antibiotics, and there is a growing concern over the transfer of antibiotic resistance genes. The MIC for 8 antibiotics was determined for 85 Bacillus species strains, Bacillus subtilis subsp. subtilis (n = 29), Bacillus licheniformis (n = 38), and Bacillus sonorensis (n = 18), all of which were isolated from starters for Sudanese bread production. All the strains were sensitive to tetracycline (8.0 mg/liter), vancomycin (4.0 mg/liter), and gentamicin (4.0 mg/liter) but resistant to streptomycin. Sensitivity to clindamycin, chloramphenicol, and kanamycin was species specific. The erythromycin resistance genes ermD and ermK were detected by PCR in all of the erythromycin-resistant (MIC, ≥16.0 mg/liter) B. licheniformis strains and one erythromycin-sensitive (MIC, 4.0 mg/liter) B. licheniformis strain. Several amino acid changes were present in the translated ermD and ermK nucleotide sequences of the erythromycin-sensitive strain, which could indicate ErmD and ErmK protein functionalities different from those of the resistance strains. The ermD and ermK genes were localized on an 11.4-kbp plasmid. All of the B. sonorensis strains harbored the bacitracin synthetase gene, bacA, and the transporter gene bcrA, which correlated with their observed resistance to bacitracin. Bacitracin was produced by all the investigated species strains (28%), as determined by ultra-high-definition quadrupole time-of-flight liquid chromatography-mass spectrometry (UHD-QTOF LC/MS). The present study has revealed species-specific variations in the antimicrobial susceptibilities of Bacillus spp. and provides new information on MIC values, as well as the occurrence of resistance genes in Bacillus spp., including the newly described species B. sonorensis.

Genotypic characterization and safety assessment of lactic acid bacteria from indigenous African fermented food products.

BACKGROUND: Indigenous fermented food products play an essential role in the diet of millions of Africans. Lactic acid bacteria (LAB) are among the predominant microbial species in African indigenous fermented food products and are used for different applications in the food and biotechnology industries. Numerous studies have described antimicrobial susceptibility profiles of LAB from different parts of the world. However, there is limited information on antimicrobial resistance profiles of LAB from Africa. The aim of this study was to characterize 33 LAB previously isolated from three different African indigenous fermented food products using (GTG)5-based rep-PCR, sequencing of the 16S rRNA gene and species-specific PCR techniques for differentiation of closely related species and further evaluate their antibiotic resistance profiles by the broth microdilution method and their haemolytic activity on sheep blood agar plates as indicators of safety traits among these bacteria. RESULTS: Using molecular biology based methods and selected phenotypic tests such as catalase reaction, CO2 production from glucose, colonies and cells morphology, the isolates were identified as Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus ghanensis, Lactobacillus plantarum, Lactobacillus salivarius, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella confusa. The bacteria were susceptible to ampicillin, chloramphenicol, clindamycin and erythromycin but resistant to vancomycin, kanamycin and streptomycin. Variable sensitivity profiles to tetracycline and gentamicin was observed among the isolates with Lb. plantarum, Lb. salivarius, W. confusa (except strain SK9-5) and Lb. fermentum strains being susceptible to tetracycline whereas Pediococcus strains and Lb. ghanensis strains were resistant. For gentamicin, Leuc. pseudomesenteroides, Lb. ghanensis and Ped. acidilactici strains were resistant to 64 mg/L whereas some W. confusa and Lb. plantarum strains had a MIC value of 16 mg/L and 32 mg/L respectively. No ß-haemolytic activity was observed, however, α-haemolytic activity was observed in 27% (9) of the strains comprising Lb. salivarius (6), W. confusa (2) and Lb. delbrueckii (1) isolates. CONCLUSIONS: The resistance to kanamycin and vancomycin is probably an intrinsic feature since similar observations were reported in the literature for LAB. Low prevalence of pathogenicity indicator traits were observed among the isolates especially with the presence of poor haemolytic activities and they could therefore be considered as interesting candidates for selection of starter cultures or probiotics for different applications.

Ancient DNA extracted from Danish aurochs (Bos primigenius): genetic diversity and preservation.

We extracted DNA from 39 Danish aurochs specimens and successfully amplified and sequenced a 252 base pair long fragment of the multivariable region I of the mitochondrial control region from 11 specimens. The sequences from these specimens dated back to 9830-2865 14Cyr BP and represent the first study of genetic variation of Danish aurochs. In addition, for all specimens we address correlations between the ability to obtain DNA sequences and various parameters such as the age of the sample, the collagen content, the museum storage period, Danish geography and whether the specimens were found in an archeological or geological context. We find that aurochs from southern Scandinavia display a star-shaped population genetic structure, that is indicative of a local and relatively recent diversification from a few ancestral haplotypes that may have originated in the ancestral Western European population before migration northwards during the retreat of the glaciers. Scenarios suggesting several invasions of genetically distinct aurochs are not supported by these analyses. Rather, our results suggest that a single continuous migration northward occurred. Our findings also suggest, although with only limited support, that aurochs in Northwestern Europe underwent a population expansion beginning shortly after the retreat of the glacial ice from Denmark and had a stable population size until the population decline that must have occurred prior to extinction. The absence of haplotypes similar to modern domestic cattle in our aurochs suggests that introgression between these species must have been limited, if it occurred at all. We found that the successful recovery of genetic material for PCR amplification correlates with sample age and local geographic conditions. However, contrary to other studies, we found no significant correlation between length of time in museum storage or the type of the locality in which a specimen was discovered (archeological or geological) and amplification success. Finally, we found large variances in our estimates of collagen content preventing an evaluation of this as an indicator of preservation quality.

Species-specific responses of Late Quaternary megafauna to climate and humans.

Despite decades of research, the roles of climate and humans in driving the dramatic extinctions of large-bodied mammals during the Late Quaternary period remain contentious. Here we use ancient DNA, species distribution models and the human fossil record to elucidate how climate and humans shaped the demographic history of woolly rhinoceros, woolly mammoth, wild horse, reindeer, bison and musk ox. We show that climate has been a major driver of population change over the past 50,000 years. However, each species responds differently to the effects of climatic shifts, habitat redistribution and human encroachment. Although climate change alone can explain the extinction of some species, such as Eurasian musk ox and woolly rhinoceros, a combination of climatic and anthropogenic effects appears to be responsible for the extinction of others, including Eurasian steppe bison and wild horse. We find no genetic signature or any distinctive range dynamics distinguishing extinct from surviving species, emphasizing the challenges associated with predicting future responses of extant mammals to climate and human-mediated habitat change.

The role of loading rate, backwashing, water and air velocities in an up-flow nitrifying tertiary filter.

The vertical distribution of nitrification performances in an up-flow biological aerated filter operated at tertiary nitrification stage is evaluated in this paper. Experimental data were collected from a semi-industrial pilot-plant under various operating conditions. The actual and the maximum nitrification rates were measured at different levels inside the up-flow biofilter. A nitrogen loading rate higher than 1.0 kg NH4-Nm(-3)_mediad(-1) is necessary to obtain nitrification activity over all the height of the biofilter. The increase in water and air velocities from 6 to 10 m h(-1) and 10 to 20 m h(-1) has increased the nitrification rate by 80% and 20% respectively. Backwashing decreases the maximum nitrification rate in the media by only 3-14%. The nitrification rate measured at a level of 0.5 m above the bottom of the filter is four times higher than the applied daily average volumetric nitrogen loading rate up to 1.5 kg NH4-N m(-3)_mediad(-1). Finally, it is shown that 58% of the available nitrification activity is mobilized in steady-state conditions while up to 100% is used under inflow-rate increase.

Heterologous expression of glycoside hydrolase family 2 and 42 ß-galactosidases of lactic acid bacteria in Lactococcus lactis.

This study characterized a glycoside hydrolase family 42 (GH42) ß-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 ß-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 ß-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 ß-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 ß-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 ß-galactosidases formed structurally similar GOS with varying preferences. The diversity of lactic acid bacteria ß-galactosidase activity in L. lactis CCE can be exploited in future nutritional or therapeutic applications.

A biofiltration model for tertiary nitrification of municipal wastewaters.

The main objective of this work concerns the evaluation of the biological aerated filtration model found in GPS-X, which had never been evaluated with adequate data. This model is interesting since it integrates the physical and biological phenomena involved during filtration with a low complexity of use. The validation of the model parameters combines experimental and theoretical approaches. Experimental data were recorded at a semi-industrial pilot scale submerged biofilter operated at a tertiary nitrification stage, receiving the effluent of a medium loaded activated sludge process for municipal wastewater. Also, several protocols were regularly applied to characterize the biofilm and the nitrogen removal performances: dry density and thickness of biofilm, nitrification rates and corresponding quantity of autotrophic biomass accumulated inside the filtering media, quantity of extracted autotrophic bacteria in the backwash water, nitrification capacity along the biofilter, as well as nitrogen compounds in the effluent. For short-term dynamic conditions, a set of reliable parameter values has been used to predict nitrogen removal for different data sets. For long-term dynamic periods, the need to adapt some of the parameters from one set of data to another is demonstrated. It is shown that the hydraulic loading rate and the backwashing frequency are the main parameters responsible for these modifications.