Optimization of airflow and aeration cycles in a new structured bed reactor configuration for carbon and nitrogen removal.

The Structured Bed Reactor with Recirculation and Intermittent Aeration (SBRRIA) is a reactor configuration that presents high efficiency of organic matter and nitrogen removal, besides low sludge production. However, operational parameters, as the recirculation rate, aeration time, and airflow, are not fully established. A bench-scale structured bed reactor with intermittent aeration was fed with synthetic effluent simulating the characteristics of sanitary sewage. The reactor was operated for 280 days with an operational hydraulic retention time (HRT) of 10 h. The reactor was operated without effluent recirculation for the first time since this approach was not yet reported, and was named Structured Bed Reactor with Intermittent Aeration (SBRIA). The COD removal was higher than 81% for all operational conditions, and the total nitrogen removal ranged from 10 to 80%. The highest efficiencies were obtained with an aeration time of 1 h 45 min (total cycle of 3 h) and an airflow rate of 4.5 L.min-1. Different nitrification and denitrification behaviours were observed, resulting in nitrification efficiencies over 90% when the reactor was submitted to higher aeration times and denitrification efficiencies above 90% when the reactor was submitted to low aeration times. The airflow ranges tested in this study affected the nitrification and the total nitrogen efficiencies. Even without effluent recirculation, the temporal profile showed that there were no peaks in the concentration of the nitrogen forms in the reactor effluent, saving electrical energy up to 75% due to pumping.

Tradição e identidade: o Ticumbi de Itaúnas em cena / Tradition and identity: the Ticumbi of Itaúnas on stage / Tradición y identidad: el Ticumbi de Itaúnas en escena

O objetivo do estudo é apresentar uma narrativa interpretativa do Ticumbi de Itaúnas, evidenciando a sua estrutura e seus significados. Para tal, realizamos um trabalho de campo tendo como método de coleta de dados a observação direta combinada com entrevistas semi-estruturadas em ocasiões da Festa de São Benedito e São Sebastião na Vila de Itaúnas/ES, nos meses de janeiro dos anos de 2018 a 2020. Compreendemos que o Ticumbi de Itaúnas é uma tradição que tem sido vivenciada e materializada dentro de um contexto comunitário e festivo que confere identidade a comunidades remanescentes quilombolas, apresentando movimentos que levam a continuidades e descontinuidades dentro da própria tradição.

The aim of the study is to present an interpretive narrative of the Ticumbi of Itaúnas, showing its structure and meanings. To this end, we carried out fieldwork using direct observation as a method of data collection combined with semi-structured interviews during the Festa de São Benedito and São Sebastião in Vila de Itaúnas/ES, from January 2018 to 2020. We understand that the Ticumbi of Itaúnas is a tradition that has been experienced and materialized within a community and festive context that gives identity to remnant quilombola communities, presenting movements that lead to continuities and discontinuities within the tradition itself.

El objetivo de la investigación es presentar una narrativa interpretativa del Ticumbi de Itaúnas, mostrando su estructura y significados. Para ello, realizamos un trabajo de campo utilizando la observación directa como método de recolección de datos combinada con entrevistas semiestructuradas durante la Festa de São Benedito y São Sebastião en Vila de Itaúnas / ES, de enero de 2018 a 2020. Entendemos que el Ticumbi de Itaúnas es una tradición que se ha vivido y materializado dentro de un contexto comunitario y festivo que da identidad a las comunidades quilombolas remanentes, presentando movimientos que conducen a continuidades y discontinuidades dentro de la propia tradición.

New Vaccine Formulations Containing a Modified Version of the Amastigote 2 Antigen and the Non-Virulent Trypanosoma cruzi CL-14 Strain Are Highly Antigenic and Protective against Leishmania infantum Challenge.

Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.

Process development for production and purification of the Schistosoma mansoni Sm14 antigen.

The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L-1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter. Mut+ transformants were selected and used in fed-batch cultivation using a 2.5L fermenter equipped with an on-line methanol control system in order to maintain constant methanol levels during induction. Optimal conditions for the expression of Sm14 by P. pastoris were found to be: dissolved oxygen at 40%, temperature of 25 °C, pH 5.0, and a constant methanol concentration of 1 gL-1. Our results show that a correctly processed Sm14 was secreted into the culture medium at levels of approximately 250  mg L-1. Sm14 from clarified culture medium was purified using a two-step procedure: anion-exchange chromatography followed by hydrophobic interaction chromatography, resulting in >95% purity with a final yield of 40% from the starting cell culture medium. This product has been tested in preliminary clinical trials and shown to elicit an antibody response with no adverse reactions.

Novel recombinant multiepitope proteins for the diagnosis of asymptomatic leishmania infantum-infected dogs.

BACKGROUND: Visceral leishmaniasis is the most severe form of leishmaniasis. Worldwide, approximately 20% of zoonotic human visceral leishmaniasis is caused by Leishmania infantum, also known as Leishmania chagasi in Latin America. Current diagnostic methods are not accurate enough to identify Leishmania-infected animals and may compromise the effectiveness of disease control. Therefore, we aimed to produce and test two recombinant multiepitope proteins as a means to improve and increase accuracy in the diagnosis of canine visceral leishmaniasis (CVL). METHODOLOGY/PRINCIPAL FINDINGS: Ten antigenic peptides were identified by CVL ELISA in previous work. In the current proposal, the coding sequences of these ten peptides were assembled into a synthetic gene. Furthermore, other twenty peptides were selected from work by our group where good B and T cell epitopes were mapped. The coding sequences of these peptides were also assembled into a synthetic gene. Both genes have been cloned and expressed in Escherichia coli, producing two multiepitope recombinant proteins, PQ10 and PQ20. These antigens have been used in CVL ELISA and were able to identify asymptomatic dogs (80%) more effectively than EIE-LVC kit, produced by Bio-Manguinhos (0%) and DPP kit (10%). Moreover, our recombinant proteins presented an early detection (before PCR) of infected dogs, with positivities ranging from 23% to 65%, depending on the phase of infection in which sera were acquired. CONCLUSIONS/SIGNIFICANCE: Our study shows that ELISA using the multiepitope proteins PQ10 and PQ20 has great potential in early CVL diagnosis. The use of these proteins in other methodologies, such as immunochromatographic tests, could be beneficial mainly for the detection of asymptomatic dogs.

Characterization of a High Affinity Phytochelatin Synthase from The Cd-Utilizing Marine Diatom Thalassiosira pseudonana.

Phytochelatin synthase (PC synthase) is the enzyme that catalyzes the production of phytochelatins, peptides of the structure (γ-Glu-Cys)n -Gly, where n = 2-11, from the sulfhydryl-containing tripeptide glutathione, in response to elevated metal exposure. Biochemical utilization of Cd in the marine diatom Thalassiosira weissfloggi, as well as unusually high ratios of PC to Cd in some Thalassiosira species including T. pseudonana Hasle et Heimdal, motivated the characterization of T. pseudonana PC synthase 1 (TpPCS1). This enzyme is the product of one of three genes in the T. pseudonana genome predicted to encode for a PC synthase based on its homology to canonical PC synthases previously examined. TpPCS1 was cloned, expressed in Escherichia coli and purified under both aerobic and anaerobic conditions. TpPCS1 exhibits several characteristics that set it distinctly apart from the well-studied PC synthase, Arabidopsis thaliana PCS1 (AtPCS1). It is extremely sensitive to oxidation, which suppresses activity, and it is readily inhibited by the addition of Cd in the absence of thiolate ligands. TpPCS1 also has significantly greater affinity for one of its key substrates, the bis-glutathionato-Cd complex. TpPCS1 kinetics is best described by a ternary complex model, as opposed to the ping-pong model used to describe AtPCS1 kinetics. The findings indicate that although the function of TpPCS1 is synonymous to that of AtPCS1, its divergent biochemistry suggests adaptation of this enzyme to the distinct trace metal chemistry of the marine environment and the unique physiological needs of T. pseudonana.

Cloning and expression of a functional core streptavidin in Pichia pastoris: strategies to increase yield.

Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min(-1) , and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L(-1) ). These parameters yielded 4.0 g L(-1) of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L(-1) of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression.

Stromal myofibroblasts in focal reactive overgrowths of the gingiva

Focal reactive overgrowths are among the most common oral mucosal lesions. The gingiva is a significant site affected by these lesions, when triggered by chronic inflammation in response to microorganisms in dental plaque. Myofibroblasts are differentiated fibroblasts that actively participate in diseases characterized by tissue fibrosis. The objective of this study was to evaluate the presence of stromal myofibroblasts in the main focal reactive overgrowths of the gingiva: focal fibrous hyperplasia (FFH), peripheral ossifying fibroma (POF), pyogenic granuloma (PG), and peripheral giant cell granuloma (PGCG). A total of 10 FFHs, 10 POFs, 10 PGs, and 10 PGCGs from archival specimens were evaluated. Samples of gingival mucosa were used as negative controls for stromal myofibroblasts. Oral squamous cell carcinoma samples, in which stromal myofibroblasts have been previously detected, were used as positive controls. Myofibroblasts were identified by immunohistochemical detection of alpha smooth muscle actin (α-sma). Myofibroblast immunostaining was qualitatively classified as negative, scanty, or dense. Differences in the presence of myofibroblasts among FFH, POF, PG, and PGCG were analyzed using the Kruskal-Wallis test. Stromal myofibroblasts were not detected in FFH, POF, PG, or PGCG. Consequently, no differences were observed in the presence of myofibroblasts among FFH, POF, PG, or PGCG (p > 0.05). In conclusion, stromal myofibroblasts were not detected in the focal reactive overgrowths of the gingiva that were evaluated, suggesting that these cells do not play a significant role in their pathogenesis.

Stromal myofibroblasts in focal reactive overgrowths of the gingiva.

Focal reactive overgrowths are among the most common oral mucosal lesions. The gingiva is a significant site affected by these lesions, when triggered by chronic inflammation in response to microorganisms in dental plaque. Myofibroblasts are differentiated fibroblasts that actively participate in diseases characterized by tissue fibrosis. The objective of this study was to evaluate the presence of stromal myofibroblasts in the main focal reactive overgrowths of the gingiva: focal fibrous hyperplasia (FFH), peripheral ossifying fibroma (POF), pyogenic granuloma (PG), and peripheral giant cell granuloma (PGCG). A total of 10 FFHs, 10 POFs, 10 PGs, and 10 PGCGs from archival specimens were evaluated. Samples of gingival mucosa were used as negative controls for stromal myofibroblasts. Oral squamous cell carcinoma samples, in which stromal myofibroblasts have been previously detected, were used as positive controls. Myofibroblasts were identified by immunohistochemical detection of alpha smooth muscle actin (α-sma). Myofibroblast immunostaining was qualitatively classified as negative, scanty, or dense. Differences in the presence of myofibroblasts among FFH, POF, PG, and PGCG were analyzed using the Kruskal-Wallis test. Stromal myofibroblasts were not detected in FFH, POF, PG, or PGCG. Consequently, no differences were observed in the presence of myofibroblasts among FFH, POF, PG, or PGCG (p > 0.05). In conclusion, stromal myofibroblasts were not detected in the focal reactive overgrowths of the gingiva that were evaluated, suggesting that these cells do not play a significant role in their pathogenesis.

Protein secretion in Pichia pastoris and advances in protein production.

Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production.

Cemental tear: a case report with nonsurgical periodontal therapy / Dilaceração cementária: relato de caso clínico com terapia periodontal não cirúrgica

PURPOSE: To report a case of cemental tear, a rare periodontal condition characterized by total or partial separation of the dental cementum, mainly addressing issues related to its diagnosis and treatment. CASE DESCRIPTION: A 50 years-old man sought dental assistance complaining of pain located in the mandibular left second premolar that showed a 4 mm probing depth with the presence of a foreign body in the distal gingival sulcus. Radiographic examination demonstrated a slight radiopaque fragment detached from the root. The fragment was removed without a periodontal flap. Histopathological examination was performed and evidenced the presence of a cementum fragment with cementum lamellae, cementocytes, and adhered periodontal ligament fibers, confirming the diagnosis of cemental tear. CONCLUSION: After a follow-up of 2 years, the nonsurgical periodontal therapy showed satisfactory clinical and radiographic outcome. Therefore, this approach should be a suitable and predictable treatment modality for the cemental tear.

OBJETIVO: Relatar um caso de dilaceração cementária, uma condição periodontal rara caracterizada pela separação total ou parcial do cemento dental, abordando principalmente aspectos relativos ao seu diagnóstico e tratamento. DESCRIÇÃO DO CASO: Um homem de 50 anos procurou assistência odontológica queixando-se de dor localizada no segundo molar inferior que apresentava profundidade de sondagem de 4 mm com presença de um corpo estranho no sulco gengival da face distal. O exame radiográfico demonstrou um fragmento radiopaco destacado da raiz. O fragmento foi removido sem cirurgia periodontal. O exame histopatológico demonstrou tratar-se de um fragmento de cemento com presença de lamelas cementárias, cementócitos e fibras do ligamento periodontal, confirmando o diagnóstico de dilaceração cementária. CONCLUSÃO: Após dois anos, o tratamento periodontal não cirúrgico demonstrou aspectos clínicos e radiográficos satisfatórios. Portanto, a terapia periodontal não cirúrgica pode ser uma modalidade de tratamento adequada e previsível para a dilaceração cementária.

Processos proliferativos não neoplásicos gengivais / Focal reactive overgrowths of the gingiva a review of the literature

A mucosa gengival pode desenvolver processos proliferativos não neoplásicos secundários ao processo inflamatório desencadeado pela ação de irritantes locais. Entre esses processos, destacam-se hiperplasia fibrosa focal, granu- loma piogênico, fibroma ossificante periférico e lesão periférica de células gigantes. 0 objetivo desta revisão foi abordar etiopatogenia, características clínicas, características histopatológicas, tratamento e prognóstico destas lesões, enfatizando a importância do conhecimento destes processos patológicos para a prática clínica odontológica.

Expression and purification of cGMP grade NY-ESO-1 for clinical trials.

NY-ESO-1 is a cancer testis antigen expressed in numerous cancers. Initial tests have shown its efficacy as a cancer vaccine, stimulating the body's own immune response against the invading tumor. To produce enough material for phase I clinical trials, a process using current good manufacturing practices to produce clinical grade material was developed and executed. His-tagged NY-ESO-1 was expressed in C41DE3 Escherichia coli under control of the T-7 promoter. NY-ESO-1 was produced in a 20 L fed-batch fermentation utilizing a pH-stat control scheme. The protein was then purified from inclusion bodies using a three-column process that achieved a yield of over 3.4 g and endotoxin below the detection limit of 0.005 EU/µg protein.

A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures.

The secreted proteome of Pichia pastoris X-33 was investigated in methanol-induced cultures with a goal to enhance the secretion and purification of recombinant proteins. In a fed-batch fermentation at 30 °C, more host proteins were found in greater concentrations compared to cultures grown at 25 °C. Protein samples collected directly from the culture media at 25 °C, as well as separated by two-dimensional (2D) gel, were subjected to ESI-MS/MS analysis. A total of 75 proteins were identified in the media from different conditions including pre- and post-methanol induction and in a strain overexpressing a recombinant schistosomiasis vaccine, Sm14-C62V. The identified proteins include native secreted proteins and some intracellular proteins, most of which have low isoelectric points (pI < 6). 2D gel analyses further revealed important characteristics, such as abundance, degradation, and glycosylation of these identified proteins in this proteome. Cell wall-associated proteins involved in cell wall biogenesis, structure, and modification comprised the majority of the secreted proteins which have been identified. Intracellular proteins such as alcohol oxidase and superoxide dismutase were also found in the proteome, suggesting some degree of cell lysis. However, both protocols show that their concentrations are significantly lower than the native secreted proteins. This study identifies proteins secreted or released into the culture media in the methanol-induced fermentation cultures of P. pastoris X-33 and suggests potential biotechnology applications based on the discovery of this proteome.

Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal.

The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L(-1). Decreasing the cultivation temperature from 25 degrees C to 16 degrees C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL(-1), and the yield of SSX2NORD was 21.6 mg L(-1).

High-level expression of a phage display-derived scFv in Pichia pastoris.

Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platforms. Once identified, antigen-binding domains on fragment antibodies or single-chain fragment antibodies (scFv) can be expressed and purified for further studies. In this chapter, we describe a method for high-level expression of a phage display-derived scFv in Pichia pastoris. The phage display-derived antibody A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for radioimmunoimaging and/or immunotherapy of human colon cancer. The expression and purification of A33scFv was optimized for the methylotrophic yeast P. pastoris. P. pastoris with a Mut(S) phenotype was selected to express A33scFv under regulation of the methanol-inducible AOX1 promoter. Here we describe a large-scale fed-batch fermentation process with an efficient online closed-loop methanol control for the production of the recombinant protein. Purification of A33scFv from clarified culture medium was done using a two-step chromatographic procedure using anion exchange and hydrophobic interaction chromatography, resulting in a final product with more than 90% purity. This chapter provides protocols that can be used as a base for process development of recombinant protein expression in P. pastoris and purification of these proteins for use in further functionality studies and in diagnostic and therapeutic applications.

Effect of bicarbonato alkalinity on gravimetric solids analysis in anaerobic wastewater treatment

La alcalinidad bicarbonática tiene papel fundamental en la estabilidad de reactores biológicos aplicados al tratamiento de aguas residuales, principalmente en sistemas anaerobios. Como algunas aguas residuales pueden sufrir severa acidificación, en algunos casos es necesaria la adición de una fuente externa de alcalinidad para que el proceso sea conducido de forma estable. En ese contexto, se evaluó el efecto de la adición de bicarbonato de sodio sobre la determinación de la concentración de sólidos. La metodología consistió en la evaluación de las concentraciones de sólidos (sólidos totales-ST, sólidos volátiles totales-SVT y sólidos fijos totales-SFT) en muestras conteniendo suero de queso y ácidos volátiles (para simulación de efluentes de reactores anaerobios). Los valores de ST, SVT y SFT fueran fuertemente influenciados, principalmente debido al aumento de los SFT. Ese efecto fue cuantificado relacionándose los valores experimentales con los teóricos, determinados por las reacciones estequiométricas de la descomposición del bicarbonato de sodio y otros compuestos formados (acetato de sodio y propionato de sodio) con el aumento de la temperatura. Así, como uno de los principales parámetros de evaluación de sistemas de tratamiento de aguas residuales es la remoción de sólidos presentes en el medio, la concentración de sólidos puede ser evaluada de forma más adecuada teniendo en cuenta la determinación de los sólidos fijos debido a las sales inorgánicas. Esa metodología es considerada adecuada cuando se adiciona grande cantidad de alcalinizada a la agua residual

Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris.

In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l(-1) in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.

Effects of feeding time and organic loading in an anaerobic sequencing batch biofilm reactor (ASBBR) treating diluted whey.

An investigation was carried out on the performance of an anaerobic sequencing batch biofilm reactor (ASBBR) treating diluted cheese whey when submitted to different feed strategies and volumetric organic loads (VOL). Polyurethane foam cubes were used as support for biomass immobilization and stirring was provided by helix impellers. The reactor with a working volume of 3 L treated 2 L of wastewater in 8-h cycles at 500 rpm and 30 degrees C. The organic loads applied were 2, 4, 8 and 12 g COD L(-1) d(-1), obtained by increasing the feed concentration. Alkalinity was supplemented at a ratio of 50% NaHCO(3)/COD. For each organic load applied three feed strategies were tested: (a) batch operation with 8-h cycle; (b) 2-h fed-batch operation followed by 6-h batch; and (c) 4-h fed-batch followed by 4-h batch. The 2-h fed-batch operation followed by 6-h batch presented the best results for the organic loads of 2 and 4 g COD L(-1) d(-1), whereas the 4-h fed-batch operation followed by 4-h batch presented results slightly inferior for the same organic loads and the best results at organic loads of 8 and 12 g COD L(-1) d(-1). The concentration of total volatile acids varied with fill time. For the higher fill times maximum concentrations were obtained at the end of the cycle. Moreover, no significant difference was detected in the maximum concentration of total volatile acids for any of the investigated conditions. However, the maximum values of propionic acid tended to decrease with increasing fill time considering the same organic load. Microbiological analyses revealed the presence of Methanosaeta-like structures and methanogenic hydrogenotrophic-like fluorescent bacilli. No Methanosarcina-like structures were observed in the samples.

Evaluation of Mut+ and MutS Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration.

Extracellular secretion of over 4 g x L(-1) of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut+ and MutS phenotypes were compared in a bioreactor under closed-loop PID control of the methanol level. As expected, the MutS phenotype has a growth rate lower than that of the Mut+ (0.37 vs 1.05 d(-1)) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut+ (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g x L(-1) for the MutS also has a positive effect on the final product concentration. Both Mut+ and MutS phenotypes reach a maximum biomass density around 450 g x L(-1) wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.