Pure Batch Research Articles

Biosurfactant assisted recovery of the C5-C11 hydrocarbon fraction from oily sludge using biosurfactant producing consortium culture of bacteria

A biosurfactant producing culture of bacteria was isolated from an automobile engine oil dump site which was later used as an inoculum in batch and continuous flow oil recovery from oily sludge. Initially, an emulsion of oily sludge was prepared by mixing 5% m/v solids: 21% v/v bituminous sludge: 77% v/v water. The isolated cultures were added to vessels with stable emulsions to facilitate the separation of oil droplets from the sludge matrix. In batches with live cultures, up to 35% oil recovery was achieved after incubation for 10 days. Further investigations were conducted in a semi-continuous feed, fed-batch plug flow reactor (FB-PFR) system. Up to 99.7% was achieved in the FB-PFR after operation for 10 days, much higher than the recovery achieved in the pure batch systems where only 35% oil was recovered after incubation for 10 days. The improved performance in the FB-PFR was attributed to differential separation of particles under variable velocity along the reactor. The culture in the reactor was predominated by Klebsiellae, Enterobacteriaceae and Bacilli throughout the experiment. A crude biosurfactant produced by the cultures was partially purified and analyzed using the liquid chromatograph coupled to a tandem mass spectrometer (LC-MS/MS) which showed that the molecular structure of the biosurfactant produced closely matched the structure of lipopeptides identified in earlier studies. This process is aimed at recovering useful oil from oily waste sludge with the added advantage of degrading aromatic organic impurities in the oil to produce a cleaner oil product. The further advantage of the FB-PFR system was that, the bacteria discharged together with effluent sludge residue further degraded chemical oxygen demand (COD) in the treated sludge thereby reducing the polluting potential of the final disposed sludge.

Abstract P3-05-10: Evaluation of HER2 expression and amplification on CTCs using DEPArray analysis and sorting followed by FISH

Abstract Introduction In metastatic breast cancer (MBC), discordant expression levels of the human epidermal growth factor receptor 2 (HER2) have been noted between primary tumors (PT) and matched metastatic lesions. Reassessment of HER2 status during treatment decisions in patients with advanced disease might help to optimize outcome. Circulating tumor cells (CTCs) offer the potential to provide a repeatedly accessible source of tumor cells for the real-time assessment of actual tumor characteristics. However, little is known on the concordance of HER2 expression on CTCs measured by immunofluorescence and the amplification status. Here we report on a preclinical study, using five spiked breast cancer cell lines, comparing semi-quantitative HER2 scoring on CellSearch (Riethdorf 2010) with objective DEPArray analysis, and subsequent FISH analysis on DEPArray-sorted tumor cells. At the moment these data are also being generated for 10 patient CTC samples. Expression and amplification status of CTCs will be compared with primary tumor tissue. Materials and methods MDA-MB-436, MCF-7, BT-20, KPL-4, and SKBR3 cells (increasing HER2 status) were spiked into donor blood and subjected to CellSearch enrichment. HER2/FITC intensity was scored manually on the CellSearch analyzer. All cell lines were injected into the DEPArray and exposure settings were optimized (FITC: exposure time 800 ms, gain 5%). These settings are further used for all preclinical and clinical samples. HER2 scoring was based on relative fluorescent units (rfu) of the HER2/FITC signal with background subtraction. Cells were sorted into pure batches of HER2 positive (DAPI+/CK+/HER2+/CD45-) and negative (DAPI+/CK+/HER2-/CD45-) tumor cells. Cytospins were formalin fixed and subjected to DAKO IQFISH. Results HER2 expression on CellSearch turned out to be very heterogeneous within the same cell line. DEPArray data was highly reproducible for all cell lines (p<0.001) and also showed a broad range of FITC rfu within the HER2 positive cell lines. Significant differences were observed between every cell line (p<0.001). The SKBR3 cell line sample also harbored a minor population of HER2- cells while this was the most positive cell line. However with FISH analysis, both HER2- and HER2+ SKBR3 cells were highly amplified (absolute HER2 count of 12-20 and HER2/CEN17 ratio of >4). MDA-MB-436 and MCF-7 cells showed no gene amplification on FISH, while in KPL-4 there was a HER2/CEN17 ratio of >2. Four patient samples with HER2 positive status on CellSearch have been run on the DEPArray. For patient 1, 1005 CTC were analyzed, 32.4% were HER2+. This was 53 (69.8% HER2+), 352 (5.7% HER2+), and 622 (6.7% HER2+) for patient 2-4 respectively. These numbers are comparable with CellSearch analysis. Discussion HER2 expression analysis by immunofluorescence is comparable between CellSearch and DEPArray, however DEPArray has the advantage that it is user-independent and highly reproducible. Furthermore, CTCs can be sorted into pure batches for downstream analysis. The FISH technique on DEPArray sorted cells is now optimized and will be used to determine the correlation between the immunofluorescent HER2 scoring and the actual amplification status of the CTCs. These data will be incorporated prior to upcoming SABCS. Citation Format: Brouwer A, Verhoest F, Vermeulen P, Rutten A, Prové A, Van Laere S, Peeters M, Dirix L. Evaluation of HER2 expression and amplification on CTCs using DEPArray analysis and sorting followed by FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-10.

Systematic Approach for Trace Level Quantification of 2-N-butyl-4-spirocyclopentane-2-imidazole-5-one Genotoxic Impurity in Irbesartan Using LC-MS/MS.

2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one has been highlighted as a potential genotoxic impurity in irbesartan. A sensitive LC-MS/MS method was developed and validated for the determination of 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one in irbesartan. Good separation between 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one and irbesartan was achieved with Symmetry C18 (100×4.6 mm, 3.5 μm) column using 65:35 v/v mixture of 0.1% formic acid and acetonitrile as mobile phase with a flow rate of 0.7 ml/min. The proposed method was specific, linear, accurate, and precise. The calibration curve shows good linearity over the concentration range of 0.1-2.0 μg/ml, which matches the range of limit of quantitation-20×limit of quantitation of estimated permitted level (1.0 μg/ml) of 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one. The method was validated as per International Conference on Harmonization guidelines and was able to quantitate 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one impurity at 1.0 μg/ml with respect to 2 mg/ml of irbesartan. 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one was not present in the three studied pure and formulation batches of irbesartan and the developed method was a good quality control tool for quantitation of 2-N-butyl-4-spirocyclopentane-2-imidazole-5-one at very low levels in irbesartan.

Cultivation of oleaginous Rhodotorula mucilaginosa in airlift bioreactor by using seawater

The enormous water resource consumption is a concern to the scale-up fermentation process, especially for those cheap fermentation commodities, such as microbial oils as the feedstock for biodiesel production. The direct cultivation of oleaginous Rhodotorula mucilaginosa in a 5-L airlift bioreactor using seawater instead of pure water led to a slightly lower biomass being achieved, at 17.2 compared to 18.1g/L, respectively. Nevertheless, a higher lipid content of 65±5% was measured in the batch using seawater as compared to the pure water batch. Both the salinity and osmotic pressure decreased as the cultivation time increased in the seawater batch, and these effects may contribute to the high tolerance for salinity. No effects were observed for the seawater on the fatty acid profiles. The major components for both batches using seawater and pure water were C16:0 (palmitic acid), C18:1 (oleic acid) and C18:2 (linoleic acid), which together accounted for over 85% of total lipids. The results of this study indicated that seawater could be a suitable option for scaling up the growth of oleaginous R.mucilaginosa, especially from the perspective of water resource utilization.

Abstract 3161: Predictive Immunotherapy Models: Overcoming the challenge of T cells gene transfer

Abstract The initiation of an appropriate T cell response is a key event in the establishment of efficient anti-tumoral response. In a context of tumor development, different T cell populations are mobilized and understanding the pathways involved in their functions and in their interactions is a major field of research in cancer immunotherapy. Thus, while the activation of antigen-specific naive CD4+ T cells that can differentiate into appropriate effector T cells and home to the tumor might be highly beneficial, regulatory CD4+ T cells are considered as deleterious. Many efforts are thus made to understand the behavior of both CD4+ T cells subsets and to decipher their functions within the tumor micro-environment. A key step to reach this goal is the development of relevant predictive cancer models including genetic modifications of T cell populations in vitro and in vivo, either for gene target studies, or to create new tools for sensitive readouts. While human and activated mouse T cells are easily transduced with lentiviral vectors, transduction of naive and regulatory murine T cells is a technical challenge since these target cells are poorly permissive to transfection or viral transduction. Here, extensive in vitro transduction data show that the use of highly concentrated (1E9 TU/mL) and pure (1E8 TU/mg of proteins) lentiviral vectors increases significantly the transduction level of naive and regulatory CD4+ T cells. Furthermore, transduced T cells exhibit an original cell phenotype without any changes of T cells specific markers expression (CD44, CD69, CD25, Ly-6C) attesting that the composition of this pure batch of lentiviral vector does not have any impact on T cells phenotype. Transduced T cells were then assessed for their properties once adoptively transferred into recipient mice. Analysis of these mice show that not only the genetically modified T cells/regulatory cells can be easily followed thanks to the fluorescent reporter but also their phenotype is not modified by the transduction, as shown in vitro. Our data bring out that the composition of lentiviral vectors in terms of titer, specific activities and purity are the success keys to ensure transduction of murine T cells. This major technological leap will allow to tightly control expression of various modifiers of these cells and thus paves the way to design original tools and develop cell-based cancer. Citation Format: Pascale Bouillé, Christine Duthoit, Régis Gayon, Vincent Feuillet, Cédric Auffray. Predictive Immunotherapy Models: Overcoming the challenge of T cells gene transfer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3161. doi:10.1158/1538-7445.AM2015-3161

Impact of nitrogen regime on fatty acid profiles of Desmodesmus quadricaudatus and Chlorella sp. and ability to produce biofuel

Abstract Microalgae have emerged as one of the most promising sources for fatty acid production. Since the various fatty acid profiles (chain length, degree of unsaturation, and branching of the chain) of the different sources influence biodiesel fuel properties, it is important to possess data on how the presence of NaNO3 as nitrogen source can influence the profile of produced fatty acids from algae. The fatty acid profiles of Desmodesmus quadricaudatus and Chlorella sp. were detected in pure batch cultures experiments. BG-11 nitrogen free medium and the medium contained 1.5 g NaNO3 l−1 were used in this investigation. At late stationary growth phase in nitrogen free medium, Chlorella sp. produced 58.39% saturated fatty acids and 41.60% unsaturated fatty acids. While in medium contained 1.5 g NaNO3 l−1Chlorella sp. produced 62.08% saturated fatty acids and 37.92% unsaturated fatty acids. In nitrogen free medium D. quadricaudatus produced 66.92% saturated fatty acids and 33.07% unsaturated fatty acids. W...

Operation of fill-and-draw reactors fed with bisphenol A as sole carbon and energy source.

While there is ample evidence in the literature that many organic xenobiotics can be biodegraded as sole carbon and energy source by pure batch cultures of selected microorganisms, the same evidence is very limited for continuous or semi-continuous mixed-culture processes simulating biological wastewater treatment plants. This study investigates for the first time whether Bisphenol A (BPA) can be removed and used as sole carbon and energy source by mixed microbial cultures in a semi-continuous process. Four fill and draw bioreactors were inoculated with unacclimated soil and operated at various hydraulic retention times (HRT), in the range of 1.7-4.2 d, using a feed composed solely of BPA (115 mg/l), and mineral salts. At steady-state, the BPA removal in the four reactors varied in the range 7.5-19% and did not show a significant trend with the HRT. The maximum growth rate on BPA was measured in the range 0.29-1.54 d-1.

Improvement of high-temperature Cr-Mo-V steel for power equipment

Methods of improving 12X1MΦ steel are considered, including electroslag remelting, vacuumarc remelting, microalloying, and the use of premium-grade pure batch (special pellets). The distinctive features of each method are described. These methods of producing steel with the required structure and purity permit increase in the long-term strength (by 15–20%), reliability, and performance of steel components.

Use of concentrated radiation for solar powered glass melting experiments

To investigate the feasibility of using concentrated solar radiation to provide process heat for glass production, a high flux solar simulator was used to melt glass forming batches. Initial experiments involved melting various glass forming batches which demonstrated that rapid and full conversion of the crystalline raw materials into an X-ray amorphous vitreous state was possible. A pure silica batch produced an X-ray amorphous product but it was not possible to refine the melt in these exploratory tests. A powdered, ternary soda–lime–silica (SLS) glass forming batch melted vigorously, with rapid gas removal, resulting in a completely transparent glass. Industrial SLS pellets were subsequently used in semi-continuous melting experiments, whereby the batch was intermittently fed into the melting zone while the beam was kept on. Additional secondary heating and insulation around outlet of the melting zone was required to achieve a semi-continuous flow of molten glass to an output crucible.

Effect of different carbon sources on the biological phosphorus removal by a sequencing batch reactor using pressurized pure oxygen

The effect of different carbon source on the efficiency of enhanced biological phosphorus removal (EBPR) from synthetic wastewater with acetate and two ratios of acetate/starch as a carbon source was investigated. Three pressurized pure oxygen sequencing batch reactor (POSBR) experiments were operated. The reactors (POSBR1, POSBR2 and POSBR3) were developed and studied at different carbon source ratios of 100% acetate, 75% acetate plus 25% starch and 50% acetate plus 50% starch, respectively. The results showed that POSBR1 had a higher phosphate release-to-uptake ratio and, respectively, in a much higher phosphorus removal efficiency (93.8%) than POSBR2 (84.7%) and POSBR3 (77.3%) within 30 days of operation. This indicated that the phosphorus removal efficiency decreased the higher the starch concentration was. It was also found that POSBR1 produced more polyhydroxyalkanoates (PHAs) than the other reactors. Based on the effect of the carbon source on the PHA concentration and consumption, the conditions of POSBR1 were favourable for the growth of polyphosphate-accumulating organisms and therefore, beneficial for the biological phosphorus removal process.

Batch production of FAEE-biodiesel using a liquid lipase formulation

The application of lipase catalysis to the production of biodiesel has received much interest during the past several years. Although most of the previous work has involved the use of immobilized enzyme, more recent work has indicated that liquid formulations of lipase can provide a highly competitive option for the conversion of oils and fats to biodiesel. This study investigates the impact of several process parameters on the production of fatty acid ethyl esters from rapeseed oil in a pure batch process on the liquid lipase formulation Callera™ Trans L. Oil conversion in excess of 98% was achieved by combining a 50% stoichiometric excess of ethanol (1.5 equivalents) with 20% (w/w) water relative to the oil. The rate of reaction was directly proportional to the amount of lipase added in this system (500–2000 LU per gram oil). Addition of glycerol to the initial reaction mixture reduced the initial reaction rate, but also improved the final yield of biodiesel by suppressing hydrolysis.

Simultaneous Determination of Two Potential Genotoxic Impurities in Zolmitriptan Using Liquid Chromatography-Tandem Mass Spectrometry

The detection and quantification of potential genotoxic impurities (PGIs) play an important role in the pharmaceutical industry. The industry needs highly selective and sensitive analytical methods for trace level quantification of these PGIs in drug substances. A sensitive and stability indicating LC-MS/MS method was developed for the simultaneous detection and quantification of (S)-4-(4-aminobenzyl)-1,3-oxazolidin-2-one (ABO) and (S)-4-(4-nitrobenzyl)- 1,3-oxazolidin-2-one (NBO) PGIs in zolmitriptan. Method utilizes, Hypersil BDS C18 column (50 mm x 4.6 mm, 3.0 μm) with electrospray ionization in selective ion recording (SIR) mode for quantitation of ABO and NBO PGIs. The method validated as per International Conference on Harmonization (ICH) guidelines and is able to quantitate ABO at 0.1 ppm and NBO at 0.2 ppm with respect to 2.0 mg/mL of zolmitriptan. The method was linear in the range of 0.1-2.0 ppm for ABO and 0.2-2.0 ppm for NBO which matches the range of LOQ-200% of specification level (1.0 ppm). The correlation coefficient obtained was >0.999 in both the cases. The impurities were not present in the three studied pure and formulation batches of zolmitriptan. The accuracy of the method was ranged between 98.6-102.5% for both ABO and NBO PGIs. The developed method is a good quality control tool to monitor ABO and NBO PGIs in zolmitriptan during its manufacture.

A Comparison of the Humoral Immune Response Induced by a Recombinant Human Protein in Wild Type Mice and in Transgenic Mice Expressing the Protein

Aim: The aim of this work was to investigate the correlation between anti drug antibody (ADA) induction and how different manufacturing processes of biopharmaceuticals affect the immunogenicity of the protein. This was done by testing four different batches of the same recombinant human protein in transgenic (Tg) mice.Methodology: Wild type (Wt) and human protein-transgenic (Tg) mice were challenged by repeated subcutaneous injections of four batches of a drug candidate protein, obtained by different purification methods. Differences between drug-specific IgG1, IgG2a, IgG2b, IgG3 and IgM antibody patterns produced in Tg vs. Wt mice were investigated and compared to the plasma cytokine profiles. A conventional ELISA was used as a reference method for ADA detection.Results: ADA responses detected in Tg mice were mainly of the IgG1 subclass and occurred only in significant response to the batch containing the highest level of proteins originating from the recombinant host cells. Wt mice, on the other hand, showed a combined IgG1/IgG2b response to all drug batches, except to the batch with the highest purity. The most pure batch failed to induce significant ADA in both Wt and Tg animals, suggesting host cell derived impurities to be a strong contributing factor to the antibody responses observed.Conclusion: Thus, an isolated IgG1 response in drug-tolerant Tg mice may serve as a potential biomarker of an immunological reaction to process-related impurities of the protein drug. In contrast, a combined IgG1/IgG2b-profile, as observed in immunoreactive Wt mice, more likely reflects a xeno-response.

Dynamic model‐based analysis of furfural and HMF detoxification by pure and mixed batch cultures of S. cerevisiae and S. stipitis

Inhibitory compounds that result from biomass hydrolysis are an obstacle to the efficient production of second-generation biofuels. Fermentative microorganisms can reduce compounds such as furfural and 5-hydroxymethyl furfural (HMF), but detoxification is accompanied by reduced growth rates and ethanol yields. In this study, we assess the effects of these furan aldehydes on pure and mixed yeast cultures consisting of a respiratory deficient mutant of Saccharomyces cerevisiae and wild-type Scheffersomyces stipitis using dynamic flux balance analysis. Uptake kinetics and stoichiometric equations for the intracellular reduction reactions associated with each inhibitor were added to genome-scale metabolic reconstructions of the two yeasts. Further modification of the S. cerevisiae metabolic network was necessary to satisfactorily predict the amount of acetate synthesized during HMF reduction. Inhibitory terms that captured the adverse effects of the furan aldehydes and their corresponding alcohols on cell growth and ethanol production were added to attain qualitative agreement with batch experiments conducted for model development and validation. When the two yeasts were co-cultured in the presence of the furan aldehydes, inoculums that reduced the synthesis of highly toxic acetate produced by S. cerevisiae yielded the highest ethanol productivities. The model described here can be used to generate optimal fermentation strategies for the simultaneous detoxification and fermentation of lignocellulosic hydrolysates by S. cerevisiae and/or S. stipitis.

A selective and sensitive UPLC–MS/MS approach for trace level quantification of four potential genotoxic impurities in zolmitriptan drug substance

The pivotal task of pharmaceutical industry is to separate and quantify the potential genotoxic impurities (PGIs) rising from the process of drug production. For trace level quantification of these PGIs we need to develop sensitive and selective analytical methods. APP, NPA, NPP and MNA have been highlighted as PGIs in zolmitriptan. A sensitive and selective UPLC–MS/MS method has developed for identification and quantification of four PGIs viz. APP, NPA, NPP and MNA in zolmitriptan. The method utilizes Hypersil BDS C8 column (50mm×4.6mm, 3.0μm) with electrospray ionization in selected ion recording (SIR) mode for quantitation of four PGIs. The method was validated as per International Conference on Harmonization (ICH) guidelines and is able to quantitate APP at 0.1ppm and NPA, NPP and MNA at 0.15ppm with respect to 5.0mg/mL of zolmitriptan. The proposed method is specific, linear, accurate and precise. The method is linear in the range of 0.1–2.0ppm for APP and 0.15–2.0ppm for NPA, NPP and MNA, which matches the range of LOQ–200% of estimated permitted level (1.0ppm). The correlation coefficient obtained was >0.999 in each case. The impurities were not present in the studied three pure and formulation batches of zolmitriptan. The accuracy of the method was ranged between 98.1 and 102.8% for four PGIs. This method is a good quality control tool for quantitation of four APP, NPA, NPP and MNA PGIs at very low levels in zolmitriptan.

5-Hydroxymethylfurfural (5-HMF) Production from Hexoses: Limits of Heterogeneous Catalysis in Hydrothermal Conditions and Potential of Concentrated Aqueous Organic Acids as Reactive Solvent System

5-Hydroxymethylfurfural (5-HMF) is an important bio-sourced intermediate, formed from carbohydrates such as glucose or fructose. The treatment at 150–250 °C of glucose or fructose in pure water and batch conditions, with catalytic amounts of most of the usual acid-basic solid catalysts, gave limited yields in 5-HMF, due mainly to the fast formation of soluble oligomers. Niobic acid, which possesses both Lewis and Brønsted acid sites, gave the highest 5-HMF yield, 28%, when high catalyst/glucose ratio is used. By contrast, we disclose in this work that the reaction of fructose in concentrated aqueous solutions of carboxylic acids, formic, acetic or lactic acids, used as reactive solvent media, leads to the selective dehydration of fructose in 5-HMF with yields up to 64% after 2 hours at 150 °C. This shows the potential of such solvent systems for the clean and easy production of 5-HMF from carbohydrates. The influence of adding solid catalysts to the carboxylic acid media was also reported, starting from glucose.

Design, Commissioning, and Start-Up of a Sequentially Fed Leach Bed Reactor Complete with an Upflow Anaerobic Sludge Blanket Digesting Grass Silage

In a wet digestion process, it is necessary to dilute high solid content feedstocks, such as grass silage. However, grass silage tends to be a problematic feedstock for wet digestion due to its tendency to float, to wrap around moving parts, and to cause inhibition to the microbial process due to production of ammonia. Grass silage may be better suited to batch digestion. However, in a batch process, half the feedstock is left behind after each cycle to provide innoculum for the next batch of feedstock. This reduces the effective reactor volume and increases capital costs. A solution is to combine the leach beds with a high-rate reactor. The system employed in this paper is termed a SLBR-UASB and is a two-phase process. The leach beds are the conduit for hydrolysis, and the methane production takes place in the UASB. The leach beds may be emptied at each cycle, reducing the size requirement of the leach beds to 67% of a pure batch system. This paper documents the problems in designing and commissioning a ...

On the nature and importance of the transition between molecules and nanocrystals: towards a chemistry of “nanoscale perfection”

This paper discusses the importance of the transition between molecular compounds and nanocrystals. The boundary between molecular and nanocrystals/nanoclusters can be defined by the emergence of the bulk phase; atoms in the core of the nanoclusters that are not bound to ligands. This transition in dimensions and structural organization is important because it overlaps with the boundary between atomically defined moieties (molecules can be isolated with increasing purity) and mixtures (nanocrystals have a distribution of sizes, shapes, and defects; they cannot be easily separated into batches of structurally identical species). Passing through this boundary, as the size of a structure increases beyond a few nanometres, the information about the position of each atom gradually disappears. This loss of structural information about a chemical structure fundamentally compromises our ability to use it as a part of a complex chemical system. If we are to engineer complex functions encoded in a chemical language, we will need pure batches of atomically defined (truly monodisperse) nanoscale compounds, and we will need to understand how to make them and preserve them over a broad range of length scales, compositions, and timeframes. In this review we survey most classes of monodisperse nanomaterials (mostly nanoclusters) and highlight the recent breakthroughs in this area which might be spearheading the development of a chemistry of "nanoscale perfection".

Co-cultivation of a bacteriocin-producing mixed culture ofBifidobacterium thermophilumRBL67 andPediococcus acidilacticiUVA1 isolated from baby faeces

We investigated the kinetics of growth, metabolism and antimicrobial activity of Bifidobacterium thermophilum RBL67 and Pediococcus acidilactici UVA1 previously isolated as a consortium from human baby faeces and producing antimicrobial proteinaceous compounds. Cell growth, antimicrobial activity, glucose consumption, organic acid production and pediocin-gene expression were monitored during pure and mixed strain batch cultures with controlled pH (6.0) at 37 degrees C. The balance of the two strains in mixed cultures was stable, yielding high cell count of 10(9) CFU ml(-1) after a very short incubation time of 4 h for UVA1 and 7 h for RBL67, and RBL67 was not affected by the high production of pediocin by UVA1 during co-culture (up to 12.8 microg ml(-1)). Furthermore, a real-time PCR assay was developed and allowed gene-expression analysis of the pediocin-gene pedA. The co-culture of RBL67 and UVA1 showed high stability, cell yields and bacteriocin production during batch cultures and has potential for use as a probiotic mixture with antibacterial properties. In addition, a gene-expression real-time PCR assay was successfully developed, used for the relative quantification of the pediocin transcript pedA and demonstrated to be a valuable complementary tool to activity assay. This is the first study reporting of a stable mixed culture of two bacteriocin-producing strains of human origin.

Factors governing performance of continuous fungal reactor during non-sterile operation – The case of a membrane bioreactor treating textile wastewater

White-rot fungi, unlike bacteria in conventional activated sludge system, can degrade wide varieties of textile dyes. Their large scale implementation, however, has been impeded due to lack of appropriate reactor system that can sustain stable performance under non-sterile environment. In this study, contrary to virtually complete decoloration of an azo dye (Acid Orange II, 100 mg L −1) in pure culture batch test, a fungal membrane bioreactor (MBR) achieved 93% removal during long-term non-sterile operation at a hydraulic retention time (HRT) of 1 d. Through a set of novel observations made in MBR and parallel batch tests, the interrelated factors responsible for incomplete dye removal, namely, bacterial disruption, fungal morphology and enzyme washout were identified. As compared to the activity of pure fungus culture, the bacteria-contaminated disintegrated MBR-sludge demonstrated low decoloration and undetectable enzymatic activity, indicating detrimental effect of bacterial contamination. Additional observations suggested close relationship between fungal morphology and enzymatic/decoloration activity under non-sterile environment. This study also demonstrated the occurrence of enzyme washout from MBR and its HRT-specific detrimental influence on removal performance. Based on the observations, certain ways to enhance decoloration were proposed.