Dynamics of sulfidogenesis associated to methanogenesis in horizontal-flow anaerobic immobilized biomass reactor

Two bench-scale horizontal anaerobic fixed bed reactors were tested to remove both sulfate and organic matter from wastewater. First, the reactors (R1 and R2) were supplied with synthetic wastewater containing sulfate and a solution of ethanol and volatile fatty acids. Subsequently, RI and R2 were fed with only ethanol or acetate, respectively. The substitution to ethanol in R1 increased the sulfate reduction efficiency from 83% to nearly 100% for a chemical oxygen demand to sulfate (COD/sulfate) ratio of 3.0. In contrast, in R2, the switch in carbon source to acetate strongly decreased sulfidogenesis and the maximum sulfate reduction achieved was 47%. Process stability in long-term experiments and high removal efficiencies of both organic matter and sulfate were achieved with ethanol as the sole carbon source. The results allow concluding that syntrophism instead of competition between the sulfate reducing bacteria and acetoclastic methanogenic archaeal populations prevailed in the reactor. (C) 2009 Elsevier Ltd. All rights reserved.

Removal of ammonium via simultaneous nitrification-denitrification nitrite-shortcut in a single packed-bed batch reactor

A polyurethane packed-bed-biofilm sequential batch reactor was fed with synthetic substrate simulating the composition of UASB reactor effluents. Two distinct ammonia nitrogen concentrations (125 and 250 mg l(-1)) were supplied during two sequential long-term experiments of 160 days each (320 total). Cycles of 24 h under intermittent aeration for periods of 1 h were applied, and ethanol was added as a carbon source at the beginning of each anoxic period. Nitrite was the main oxidized nitrogen compound which accumulated only during the aerated phases of the batch cycle. A consistent decrease of nitrite concentration started always immediately after the interruption of oxygen supply and addition of the electron donor. Removal to below detection limits of all nitrogen soluble forms was always observed at the end of the 24 h cycles for both initial concentrations. Polyurethane packed-bed matrices and ethanol amendments conferred high process stability. Microbial investigation by cloning suggested that nitrification was carried out by Nitrosomonas-like species whereas denitrification was mediated by unclassified species commonly observed in denitrifying environments. The packed-bed batch bioreactor favored the simultaneous colonization of distinct microbial groups within the immobilized microbial biomass. The biofilm was capable of actively oxidizing ammonium and denitrification at high ratios in intermittent intervals within 24 h cycles. (c) 2008 Elsevier Ltd. All rights reserved.

Performance of a horizontal-flow anaerobic immobilized biomass (HAIB) reactor and dynamics of the microbial community during degradation of pentachlorophenol (PCP)

The anaerobic biological treatment of pentachlorophenol (PCP) and methanol as the main carbon source was investigated in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor at 30 +/- 1 degrees C, during a 220-day trial period. The reactor biomass was developed as an attached biofilm on polyurethane foam particles, with 24 h of hydraulic retention time. The PCP concentrations, which ranged from 2.0 to 13.0 mg/L, were controlled by adding synthetic substrate. The HAIB reactor reduced 97% of COD and removed 99% of PCP. The microbial biofilm communities of the HAIB reactor amended with PCP, without previous acclimatization, were characterized by polymerase chain reaction (PCR) and amplified ribosomal DNA restriction analysis (ARDRA) with specific Archaea oligonucleotide primers. The ARDRA technique provided an adequate analysis of the community, revealing the profile of the selected population along the reactor. The biomass activities in the HAIB reactor at the end of the experiments indicated the development of PCP degraders and the maintenance of the population of methanogenic Archaea, ensuring the high efficiency of the system treating PCP with added methanol as the cosubstrate. The use of the simplified ARDRA method enabled us to monitor the microbial population with the addition of high concentrations of toxic compounds and highlighting a selection of microorganisms in the biofilm. (C) 2008 Published by Elsevier Ltd.

Evaluation of the microbial diversity in a horizontal-flow anaerobic immobilized biomass reactor treating linear alkylbenzene sulfonate

The purpose of this work was to assess the degradation of linear alkylbenzene sulfonate (LAS) in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor. The reactor was filled with polyurethane foam where the sludge from a sanitary sewage treatment was immobilized. The hydraulic detention time (HDT) used in the experiments was of 12 h. The reactor was fed with synthetic substrate (410 mg l(-1) of meat extract, 115 mg l(-1) of starch, 80 mg l(-1) of saccharose, 320 mg l(-1) of sodium bicarbonate and 5 ml l(-1)of salt solution) in the following stages of operation: SI-synthetic substrate, SII-synthetic substrate with 7 mg l(-1) of LAS, SIII-synthetic substrate with 14 mg l(-1) of LAS and SIV-synthetic substrate containing yeast extract (substituting meat extract) and 14 mg l(-1) of LAS, without starch. At the end of the experiment (313 days) a degradation of similar to 35% of LAS was achieved. The higher the concentration of LAS, the greater the amount of foam for its adsorption. This is necessary because the isotherm of LAS adsorption in the foam is linear for the studied concentrations (2 to 50 mg l(-1)). Microscopic analyses of the biofilm revealed diverse microbial morphologies, while Denaturing Gradient Gel Eletrophoresis (DGGE) profiling showed variations in the population of total bacteria and sulphate-reducing bacteria (SRB). The 16S rRNA gene sequencing and phylogenetic analyses revealed that the members of the order Clostridiales were the major components of the bacterial community in the last reactor operation step.

Influence of organic shock loads in an ASBBR treating synthetic wastewater with different concentration levels

Safe application of the anaerobic sequencing biofilm batch reactor (ASBBR) still depends on deeper insight into its behavior when faced with common operational problems in wastewater treatments such as tolerance to abrupt variations in influent concentration, so called shock loads. To this end the current work shows the effect of organic shock loads on the performance of an ASBBR, with a useful volume of 5 L, containing 0.5-cm polyurethane cubes and operating at 30 degrees C with mechanical stirring of 500 rpm. In the assays 2 L of two types of synthetic wastewater were treated in 8-h cycles. Synthetic wastewater I was based on sucrose-amide-cellulose with concentration of 500 mg COD/L and synthetic wastewater II was based on volatile acids with concentration ranging from 500 to 2000 mg COD/L. Organic shock loads of 2-4 times the operation concentration were applied during one and two cycles. System efficiency was monitored before and after application of the perturbation. When operating with concentrations from 500 to 1000 mg COD/L and shock loads of 2-4 times the influent concentration during one or two cycles the system was able to regain stability after one cycle and the values of organic matter, total and intermediate volatile acids, bicarbonate alkalinity and pH were similar to those prior to the perturbations. At a concentration of 2000 mg COD/L the reactor appeared to be robust, regaining removal efficiencies similar to those prior to perturbation at shock loads twice the operation concentration lasting one cycle and stability was recovered after two cycles. However, for shock loads twice the operation concentration during two cycles and shock loads four times the operation concentration during one or two cycles filtered sample removal efficiency decreased to levels different from those prior to perturbation, on an average of 90-80%, approximately, yet the system managed to attain stability within two cycles after shock application. Therefore, this investigation envisions the potential of full scale application of this type of bioreactor which showed robustness to organic shock loads, despite discontinuous operation and the short times available for treating total wastewater volume. (c) 2007 Elsevier Ltd. All rights reserved.

Degradation of detergent (linear alkylbenzene sulfonate) in an anaerobic stirred sequencing-batch reactor containing granular biomass

This study aimed to determine the efficiency of an anaerobic stirred sequencing-batch reactor containing granular biomass for the degradation of linear alkylbenzene sulfonate (LAS), a surfactant present in household detergent. The bioreactor was monitored for LAS concentrations in the influent, effluent and sludge, pH, chemical oxygen demand, bicarbonate alkalinity, total solids, and volatile solids. The degradation of LAS was found to be higher in the absence of co-substrates (53%) than in their presence (24-37%). Using the polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE), we identified populations of microorganisms from the Bacteria and Archaea domains. Among the bacteria, we identified uncultivated populations of Arcanobacterium spp. (94%) and Opitutus spp. (96%). Among the Archaea, we identified Methanospirillum spp. (90%), Methanosaeta spp. (98%), and Methanobacterium spp. (96%). The presence of methanogenic microorganisms shows that LAS did not inhibit anaerobic digestion. Sampling at the last stage of reactor operation recovered 61 clones belonging to the domain bacteria. These represented a variety of phyla: 34% shared significant homology with Bacteroidetes, 18% with Proteobacteria, 11% with Verrucomicrobia, 8% with Fibrobacteres, 2% with Acidobacteria, 3% with Chlorobi and Firmicutes, and 1% with Acidobacteres and Chloroflexi. A small fraction of the clones (13%) were not related to any phylum. Published by Elsevier Ltd.

Application of molecular techniques to evaluate the methanogenic archaea and anaerobic bacteria in the presence of oxygen with different COD : sulfate ratios in a UASB reactor

In this paper, the microbial characteristics of the granular sludge in the presence of oxygen (3.0 +/- 0.7 mg O-2 1(-1)) were analyzed using molecular biology techniques. The granules were provided by an upflow anaerobic sludge blanket (UASB) operated over 469 days and fed with synthetic substrate. Ethanol and sulfate were added to obtain different COD/SO42- ratios (3.0, 2.0, and 1.6). The results of fluorescent in situ hybridization (FISH) analyses showed that archaeal cells, detected by the ARC915 probe, accounted for 77%, 84%, and 75% in the COD/SO42- ratios (3.0, 2.0, and 1.6, respectively). Methanosaeta sp. was the predominant acetoclastic archaea observed by optical microscopy and FISH analyses, and confirmed by sequencing of the excised bands of the DGGE gel with a similarity of 96%. The sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris (similarity of 99%) was verified by sequencing of the DGGE band. Others identified microorganism were similar to Shewanella sp. and Desulfitobacterium hafniense, with similarities of 95% and 99%, respectively. These results confirmed that the presence of oxygen did not severely affect the metabolism of microorganisms that are commonly considered strictly anaerobic. We obtained mean efficiencies of organic matter conversion and sulfate reducing higher than 74%. (C) 2008 Elsevier Ltd. All rights reserved.

Biodeterioration of painted mortar surfaces in tropical urban and coastal situations: Comparison of four paint formulations

Four different architectural acrylic paint formulations were tested by exposure to weathering for 7 years in the urban site of Sao Paulo and the coastal site of Ubatuba, South-East Brazil. Surface discolorations and detachment of coatings were assessed and the components of the biofilms were identified by standard microbiological methods. The painted surfaces of the mortar panels were much more discolored in Ubatuba, where major components of the biofilms were the cyanobacteria Gloeocapsa and Scytonema. In two of the four paint films, a pink coloration on the surface at this coastal site, caused mainly by red-pigmented Gloeocapsa, produced high discoloration ratings, but low degradation (as measured by detachment). Biofilms in Sao Paulo contained the same range of phototrophs, but in lesser quantity. However, fungal numbers, as determined by plating, were higher. Detachment ratings in this urban site were only slightly lower than in Ubatuba. The matt paint performed worst of the four, with silk and semi-gloss finishes giving lowest biodeterioration ratings. The matt elastomeric paint performed well at both sites, apart from becoming almost 100% covered by the pink biofilm in Ubatuba. Unpainted mortar panels became intensely discolored with a black biofilm, showing that all the paints had achieved one of their objectives, that of surface protection of the substrate. The value of PVC (pigment volume content) as an indicator of coatings biosusceptibility, is questioned. (C) 2011 Elsevier Ltd. All rights reserved.

Evaluation of the effect of ammonium carbamate on the stability of proteins

BACKGROUND: The use of the volatile salt ammonium carbamate in protein downstream processing has recently been proposed. The main advantage of using volatile salts is that they can be removed from precipitates and liquid effluents through pressure reduction or temperature increase. Although previous studies showed that ammonium carbamate is efficient as a precipitant agent, there was evidence of denaturation in some enzymes. In this work, the effect of ammonium carbamate on the stability of five enzymes was evaluated. RESULTS: Activity assays showed that alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), lysozyme (1,4-beta-N-acetylmuramoylhydrolase, EC 3.2.1.17) and lipase (triacyl glycerol acyl hydrolase, EC 3.1.1.3) did not undergo activity loss in ammonium carbamate solutions with concentrations from 1.0 to 5.0 mol kg(-1), whereas cellulase complex (1,4-(1,3 : 14)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) and peroxidase (hydrogen peroxide oxidoreductase, EC 1.11.1.7) showed an average activity loss of 55% and 44%, respectively. Precipitation assays did not show enzyme denaturation or phase separation for alpha-amylase and lipase, while celullase and peroxidase precipitated with some activity reduction. Analysis of similar experiments with ammonium and sodium sulfate did not affect the activity of enzymes. CONCLUSION: Celullase and peroxidase were denatured by ammonium carbamate. While more systematic studies are not available, care must be taken in designing a protein precipitation with this salt. The results suggest that the generally accepted idea that salts that denature proteins tend to solubilize them does not hold for ammonium carbamate. (C) 2010 Society of Chemical Industry

Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h(-1), which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO(2) production and O(2) consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained similar to 1.5-3 x 10(7) cells/mL after 3-4 clays of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 mu g/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 mu g/10(7) cells at clays 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 mu g/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function. (C) 2010 Elsevier B.V. All rights reserved.

Precipitation of Porcine Insulin With Carbon Dioxide

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO(2)) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO(2) as an acid- precipitating agent. The Solubility of porcine insulin in NaHCO(3) solutions in pressurized CO(2) was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoclectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self-association reactions were also taken into account. The biological activity of insulin was maintained after precipitation With CO(2), although some activity can be lost if foam is formed in the depressurization step. Biotechnol. Bioeng. 2009;103: 909-919. (C) 2009 Wiley Periodicals, Inc.

Retamycin production by immobilized cells of Streptomyces olindensis ICB20 in repeated-batch cultures

Repeated-batch cultures of Ca-alginate immobilized cells of Streptomyces olindensis ICB20 for retamycin production were carried out in two different bioreactors: a basket-type stirred tank reactor (BSTR) and a bubble column reactor (BCR). Higher average values of retamycin content (R) and productivity (P-R) were achieved in the BSTR cultures (about 1.7 AU and 0.031 AU h(-1), respectively) compared to those obtained in the BCR cultures (about 0.6 AU and 0.012 AU h(-1), respectively). The BCR, on the other hand, presented significantly better operation stability than the BSTR, which makes the former much more promising regarding future industrial applications. (C) 2008 Elsevier Ltd. All rights reserved.

Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (A degrees C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 A degrees C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 x 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (similar to 80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (similar to 9 x 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce similar to 1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L-1, was verified in the same culture while the highest concentration, 55 mg L-1, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.