Enzymatic hydrolysis of floatable fatty wastes from dairy and meat food-processing industries and further anaerobic digestion.

The wastewater from food-processing industries is generally heavily charged with lipids and proteins. Flotation process is commonly applied to separate the hydrophobic material phase, producing flotation froth, a waste that has high levels of fats and proteins. Enzymatic hydrolysis may be used to overcome the difficulty of fat biotransformation in a subsequent anaerobic digestion. In the present work, wastes from the flotation process of two industries (dairy and poultry slaughterhouse) were hydrolyzed with a commercial lipase and without enzyme addition (control). The effect of adjusting the pH at the beginning of the hydrolytic assays was also investigated. The long chain free fatty acids (LCFAs) released were identified and quantified and 5-d digestion assays were conducted with the hydrolyzed material. The results indicated that the hydrolysis assays conducted with initial pH adjusted to 7.0 and the utilization of a commercial enzyme promoted a higher increase in amounts of LCFAs, particularly of unsaturated acids. In most anaerobic digestion assays, the specific methane production showed a decreasing trend with the increase of unsaturated fatty acids in the medium. In general, the utilization of a commercial enzyme (lipase) in the hydrolysis process did not contribute to enhancing methane production in 5-d anaerobic digestion assays.

Treatment of petroleum refinery wastewater containing heavily polluting substances in an aerobic submerged fixed-bed reactor.

Petroleum refineries produce large amount of wastewaters, which often contain a wide range of different compounds. Some of these constituents may be recalcitrant and therefore difficult to be treated biologically. This study evaluated the capability of an aerobic submerged fixed-bed reactor (ASFBR) containing a corrugated PVC support material for biofilm attachment to treat a complex and high-strength organic wastewater coming from a petroleum refinery. The reactor operation was divided into five experimental runs which lasted more than 250 days. During the reactor operation, the applied volumetric organic load was varied within the range of 0.5-2.4 kgCOD.m(-3).d(-1). Despite the inherent fluctuations on the characteristics of the complex wastewater and the slight decrease in the reactor performance when the influent organic load was increased, the ASFBR showed good stability and allowed to reach chemical oxygen demand, dissolved organic carbon and total suspended solids removals up to 91%, 90% and 92%, respectively. Appreciable ammonium removal was obtained (around 90%). Some challenging aspects of reactor operation such as biofilm quantification and important biofilm constituents (e.g. polysaccharides (PS) and proteins (PT)) were also addressed in this work. Average PS/volatile attached solids (VAS) and PT/VAS ratios were around 6% and 50%, respectively. The support material promoted biofilm attachment without appreciable loss of solids and allowed long-term operation without clogging. Microscopic observations of the microbial community revealed great diversity of higher organisms, such as protozoa and rotifers, suggesting that toxic compounds found in the wastewater were possibly removed in the biofilm.

Application of ozonation to reduce biological sludge production in an industrial wastewater treatment plant.

Biosolids production in the activated sludge process generates an additional cost to wastewater treatment plants due to the growing requirements for sludge treatment and disposal. This work focuses on the application of ozonation to reduce sludge production in an industrial wastewater treatment plant. The results show that ozonation was able to promote cell wall rupture, releasing intracellular matter into the liquid medium. This effect was observed by the increase in concentrations of DNA (1.14 to 7.83 mg/L) and proteins (0.5 to 45.602 mg/L) in the liquid phase, when ozonation was applied during 10 min, using 30 mg/L of ozone. Reduction of sludge production was assessed by calculating the observed sludge yield coefficient (Y) in bench-scale continuous experiments conducted with varying proportions of ozonated sludge in the recycle stream and recycle ratios. Reduction of sludge production ranged from 14 to 39%, depending on the experimental conditions. The best result in terms of sludge excess reduction was achieved when 20% of the recycle sludge was ozonated and the recycle ratio was 0.67.

Heavy metal removal from synthetic wastewaters in an anaerobic bioreactor using stillage from ethanol distilleries as a carbon source.

This work was conducted to investigate the possibility of using stillage from ethanol distilleries as substrate for sulfate reducing bacteria (SRB) growth and to evaluate the removal efficiency of heavy metals present in wastewaters containing sulfates. The experiments were carried out in a continuous bench-scale Upflow Anaerobic Sludge Blanket reactor (13 l) operated with a hydraulic retention time of 18 h. The bioreactor was inoculated with 7 l of anaerobic sludge. Afterwards, an enrichment procedure to increase SRB numbers was started. After this, cadmium and zinc were added to the synthetic wastewater, and their removal as metal sulfide was evaluated. The synthetic wastewater used represented the drainage from a dam of a metallurgical industry to which a carbon source (stillage) was added. The results showed that high percentages of removal (>99%) of Cd and Zn were attained in the bioreactor, and that the removal as sulfide precipitates was not the only form of metal removal occurring in the bioreactor environment.

Toxicity evaluation of the process effluent streams of a petrochemical industry.

The physico-chemical characteristics and the acute toxicity of several wastewater streams, generated in the industrial production of synthetic rubber, were determined. The acute toxicity was evaluated in bioassays using different organisms: Danio rerio (fish), Lactuca sativa (lettuce) and Brachionus calyciflorus (rotifer). The removal of toxicity attained in the industrial wastewater treatment plant was also determined upstream and downstream of the activated sludge process. The results obtained indicate that the critical streams in terms of acute toxicity are the effluents from the liquid polymer unit and the spent caustic butadiene washing stage. The biological treatment was able to partially remove the toxicity of the industrial wastewater. However, a residual toxicity level persisted in the biotreated wastewater. The results obtained with Lactuca sativa showed a high degree of reproducibility, using root length or germination index as evaluation parameters. The effect of volatile pollutants on the toxicity results obtained with lettuce seeds was assessed, using ethanol as a model compound. Modifications on the assay procedure were proposed. A strong correlation between the toxic responses of Lactuca sativa and Danio rerio was observed for most industrial effluent streams.

New group-specific 16S rDNA primers for monitoring foaming mycolata during saline waste-water treatment.

Newly designed group-specific PCR primers for denaturing gradient gel electrophoresis (DGGE) were used to investigate foaming mycolata from a bioreactor treating an industrial saline waste-water. Genetic profiles on DGGE gels were different with NaCl at 1.65 and 8.24 g l(-1), demonstrating that mycolata community was affected by salinity. A semi-nested PCR strategy resulted in more bands in community genetic profiles than direct amplification. DNA sequencing of bands confirmed the efficacy of the novel primers with sequences recovered being most similar to foam producing mycolata. The new group-specific primers/DGGE approach is a new step toward a more complete understanding of functionally important groups of bacteria involved in biological treatment of waste-water.

Treatment of petroleum refinery sourwater by advanced oxidation processes.

The performance of several oxidation processes to remove organic pollutants from sourwater was investigated. Sourwater is a specific stream of petroleum refineries, which contains slowly biodegradable compounds and toxic substances that impair the industrial biological wastewater treatment system. Preliminary experiments were conducted, using the following processes: H2O2, H2O2/UV, UV, photocatalysis, ozonation, Fenton and photo-Fenton. All processes, except Fenton and photo-Fenton, did not lead to satisfactory results, reducing at most 35% of the sourwater dissolved organic carbon (DOC). Thus, further experiments were performed with these two techniques to evaluate process conditions and organic matter removal kinetics. Batch experiments revealed that the Fenton reaction is very fast and reaches, in a few minutes, an ultimate DOC removal of 13-27%, due to the formation of iron complexes. Radiation for an additional period of 60 min can increase DOC removal up to 87%. Experiments were also conducted in a continuous mode, operating one 0.4L Fenton stirred reactor and one 1.6L photo-Fenton reactor in series. DOC removals above 75% were reached, when the reaction system was operated with hydraulic retention times (HRT) higher than 85 min. An empirical mathematical model was proposed to represent the DOC removal kinetics, allowing predicting process performance quite satisfactorily.

Phenol removal from high salinity effluents using Fenton's reagent and photo-Fenton reactions.

The removal of pollutants in saline medium by the Fenton's reagent needs a more detailed investigation, since the presence of chloride may inhibit or retard degradation. Phenol was used as a model pollutant and the influence of some important process variables for the removal of total organic carbon and phenol were investigated, such as FeSO4 and H2O2 concentrations, pH and salinity. The reactivity of iron cations and alternative procedures of applying UV radiation (photo-Fenton) were evaluated. Phenol was fast and completely removed by the Fenton's process even in a high saline medium (50,000mg NaCll(-1)). However, TOC was only moderately or poorly removed in saline media, depending on the salt concentration. When the photo-Fenton process was used, mineralization was improved and high TOC removals were observed in moderately saline media (NaCl concentration < or =10,000mgl(-1)). For the highest NaCl concentration tested (50,000mgl(-1)) only a moderate TOC removal was observed (50%).

Treatment and detoxification of a sanitary landfill leachate.

The leachate from an old sanitary landfill (Gramacho Metropolitan Landfill, Rio de Janeiro) was characterized and submitted to coagulation and flocculation treatment followed by ozonation and ammonia stripping. The performance of the treatment was assessed by monitoring the removal of organic matter (COD and TOC), ammonium nitrogen and metals. Detoxification was assessed by determining acute toxicity, using the following organisms: Vibrio fisheri, Daphnia similis, Artemia salina and Brachydanio rerio. Membrane fractionation was employed to infer the range of molecular masses of the pollutants found in the effluent, as well as the toxicity associated to these fractions. Of the techniques under investigation, coagulation and flocculation followed by ammonia stripping were the most effective for toxicity and ammonium nitrogen removal. Membrane fractionation was effective for COD removal; however, acute toxicity was almost the same in all the fractionated samples. Ozonation was moderately effective for COD removal, but significant toxicity removal was only attained when high ozone doses were used.

Extraction of activated sludge bacteria exopolymers by ultrasonication.

Ultrasonication for the extraction of activated sludge exopolymers was evaluated by total cell count, exopolymer extraction and transmission electron microscopy (TEM). A high deflocculation was achieved after 30 s of sonication in PBS (phosphate-buffered saline). TEM showed that cell lysis was minimal only when sludges were sonicated for 30 s. For sludges sonicated for 30, 90 and 420 s and stained with Ruthenium Red, exopolymers were not extracted on a large scale without considerable cell lysis. Sludges sonicated for 30 s in EDTA gave a larger fraction of damaged cells and also showed copious amounts of attached exopolymers.

Flotation techniques for oily water treatment.

The aim of this work is to develop alternative techniques for the treatment of waters heavily contaminated by oil. Furthermore, the treatment system should achieve high removal efficiencies even under unfavorable conditions, when oil is finely dispersed in the water phase and oil droplet sizes range from 5-30 mm and concentrations are between 100 and 300 mg l(-1). The first experiments were carried out in an induced air flotation system where a flotation column performance was evaluated in batch and continuous operation. The second system investigated in this work focused on the association of centrifugal separation in a hydrocyclone and the flotation operation. The considered system is characterized by the association of these two processes, resulting the set-up in a compact unit (centrifugal flotation system). The bubbles generation and the droplet-bubble contact are performed through a gas-liquid ejector, while the separation of the phase rich in oil (froth phase) is promoted by a hydrocyclone. In both systems, it was possible to reduce the chemical demand of oxygen by more than 85% and the oil concentration of an emulsion containing droplets with sizes between 10 and 20 mm, using Polyacrilamide as destabilizing agent.

Oilfield wastewater treatment by combined microfiltration and biological processes.

This work deals with the treatment of offshore oilfield wastewater from the Campos Basin (Rio de Janeiro State, Brazil). After coarse filtration, this high saline wastewater was microfiltrated through mixed cellulose ester (MCE) membranes, resulting in average removals of COD, TOC, O&G and phenols of 35%, 25%, 92% and 35%, respectively. The permeate effluent was fed into a 1-L air-lift reactor containing polystyrene particles of 2mm diameter, used as support material. This reactor was operated for 210 days, at three hydraulic retention times (HRT): 48, 24 and 12h. Even when operated at the lowest HRT (12 h), removal efficiencies of 65% COD, 80% TOC, 65% phenols and 40% ammonium were attained. The final effluent presented COD and TOC values of 230 and 55 mg/L, respectively. Results obtained by gas chromatography analyses and toxicity tests with Artemia salina showed that a significant improvement in the effluent's quality was achieved after treatment by the combined (microfiltration/biological) process.

Laccase from Trametes versicolor: stability at temperature and alkaline conditions and its effect on biobleaching of hardwood kraft pulp.

The enzyme laccase was produced by the white-rot fungus Trametes versicolor in repeated batches cultures with immobilized mycelium. Two different culture conditions were used. Enzymes produced were evaluated regarding their stability at high temperatures (55 degrees C and 65 degrees C) and at alkaline conditions (pH 7.0 and pH 8.0) having in view the application of these enzymes in biobleaching of hardwood Kraft pulp. Biobleaching experiments were divided in two parts, enzymatic prebleaching followed by chemical bleaching. In the enzymatic prebleaching the enzyme laccase was used at two conditions of pH and temperature, whereas the reaction time was fixed at 1 h in all pretreatments. In the chemical bleaching the DEDED and DEpDED sequences were used. The enzyme action was evaluated by Kappa number, viscosity, and brightness at the end of bleaching sequences. There were obtained values of Kappa numbers lower than control assays, viscosities compatible with industrial pulps, and brightness higher than controls, when pulps were pretreated for 1 h with laccase at pH 8.0 and 55 degrees C.

Enzymatic synthesis of medium-chain triglycerides in a solvent-free system.

The synthesis of tricaprylin, tricaprin, trilaurin, and trimyristin in a solvent- free system was conducted by mixing a commercial immobilized lipase (Lipozyme IM 20, Novo Nordisk, Bagsvaerd, Denmark) with the organic reactants (glycerol and fatty acids) in a 20-mL batch reactor with constant stirring. In a first set of experiments, the effect of water concentration (0-6%) on the reaction conversion was shown to be negligible. In a second set of experiments, the effects of temperature (70-90 degrees C), fatty acid/glycerol molar ratio (1-5), and enzyme concentration (1-9% [w/w]) on the reaction conversion were determined by the application of a 3 x 3 experimental design. The reactions were carried out for 26 h and the nonpolar phase was analyzed by gas chromatography (GC). Appreciable levels of medium-chain triglycerides were achieved, except for tricaprylin. For the triglyceride production, higher selectivity was attained under the following conditions: molar ratio of 5, enzyme concentration of 5 or 9% (w/w) and temperatures of 70 degrees C (tricaprin), 80 degrees C (trilaurin), and 90 degrees C (trimyristin). Statistical analysis indicated that the fatty acid/glycerol molar ratio was the most significant variable affecting the synthesis of triglycerides.

Mathematical modeling of lipase and protease production by Penicillium restrictum in a batch fermenter.

This work presents a mathematical model that describes time course variations of extracellular lipase and protease activities for the batch fermentation of the fungus Penicillium restrictum, a new and promising strain isolated from soil and wastes of a Brazilian babassu coconut oil industry. The fermentation process was modeled by an unstructured model, which considered the following dependent variables: cells, fat acid, dissolved oxygen concentrations, lipase and protease activities, and cell lysate concentration. The last variable represents the amount of cells that has been lysed by the shear stress and natural cell death. Proteases released to the medium, as consequence of this process, enhance lipase inactivation. The model is able to predict the effects of some operation variables such as air flow rate and agitation speed. The mathematical model was validated against batch-fermentation data obtained under several operating conditions. Because substrate concentration has antagonistic effects on lipase activity, a typical optimization scheme should be developed in order to minimize these deleterious effects while maximizing lipase activity.

Ethanol from babassu coconut starch. Technical and economical aspects.

This study describes a pioneering industrial-scale experience by Tobasa in ethanol production from the amylaceous flour obtained by mechanical processing of the babassu mesocarp. Technical aspects related to enzymatic and fermentation processes, as well as overall economical aspects, are discussed. When produced in a small-size industrial plant (5000 L/d), babassu ethanol has a final cost of about $218/m3. The impact of raw materials, production, and processing (enzymes, steam, energy, and so on) on the final product cost is also presented. Babassu coconut ethanol can be produced at low cost, compared with traditional starchy raw materials or sugar cane. The net profitability of ethanol production is about 40% for babassu coconut and just 10% for sugar cane. If the estimated renewable babassu resources were entirely industrially used, 1 billion L/yr of ethanol could be produced, which would roughly correspond to 8% of the current Brazilian ethanol production.

Production and characterization of Phanerochaete chrysosporium lignin peroxidases for pulp bleaching.

The production of lignin peroxidase from Phanerochaete chrysosporium was studied using immobilized mycelia in nylon-web cubes in semicontinuous fermentation using glucose pulses or ammonium tartrate pulses. Consistent enzyme production was achieved when glucose pulses were used, leading to an average activity of 253 U/L. The crude enzyme was added to eucalyptus kraft pulp before conventional and ECF bleaching sequences. Optimization of the enzymatic pretreatment led to the following operational conditions: enzyme load of 2 U/g of pulp, hydrogen peroxide addition rate of 10 ppm/h, and reaction time of 60 min. Pulp final characteristics were dependent on the chemical treatment sequence that followed enzymatic pretreatment. The chief advantage of enzymatic pretreatment was pulp viscosity preservation, which was observed in most of the experiments carried out with seven different chemical treatment sequences.

A stable lipase from Candida lipolytica: cultivation conditions and crude enzyme characteristics.

Although lipases have been intensively studied, some aspects of enzyme production like substrate uptake, catabolite repression, and enzyme stability under long storage periods are seldom discussed in the literature. This work deals with the production of lipase by a new selected strain of Candida lipolytica. Concerning nutrition, it was observed that inorganic nitrogen sources were not as effective as peptone, and that oleic acid or triacylglycerides (TAG) were essential carbon sources. Repression by glucose and stimulation by oleic acid and long chain TAG (triolein and olive oil) were observed. Extracellular lipase activity was only observed at high levels at late stationary phase, whereas intracellular lipase levels were constant and almost undetectable during the cultivation period, suggesting that the produced enzyme was attached to the cell wall, mainly at the beginning of cultivation. The crude lipase produced by this yeast strain shows the following optima conditions: pH 8.0-10.0, temperature of 55 degrees C. Moreover, this preparation maintains its full activity for at least 370 d at 5 degrees C.

Lipase production by Penicillium restrictum in a bench-scale fermenter : effect of carbon and nitrogen nutrition, agitation, and aeration.

A preliminary screening work selected Penicillium restrictum as a promising micro-organism for lipase production. The physiological response of the fungus towards cell growth and enzyme production upon variable carbon and nitrogen nutrition, specific air flow rate (Qa) and agitation (N) was evaluated in a 5-L bench-scale fermenter. In optimized conditions for lipase production meat peptone at 2% (w/v) and olive oil at 1% (w/v) were used in a growth medium with a C/N ratio of 9.9. Higher C/N ratios favored cell growth in detriment of enzyme production. Low extracellular lipase activities were observed using glucose as carbon source suggesting glucose regulation. Final lipase accumulation of 13,000 U/L was obtained, using optimized specific air flow rate (Qa) of 0.5 wm and an impeller speed (N) of 200 rpm. Agitation showed to be an important parameter to ensure nutrient availability in a growth medium having olive oil as carbon source.

Biological treatment of a synthetic gold milling effluent.

This paper reports on biological sludge acclimatisation and the results concerning the removal of free cyanide, thiocyanate and metallocyanides (copper, iron and zinc) from a synthetic gold milling effluent. The experiments were carried out in a continuous bench-scale bioreactor, and the experimental set-up consisted of two identical units, one of which served as control. The acclimatisation of the biomass was based on a stepwise procedure, in which the proportion of synthetic solution in the influent was gradually increased. The reactors were fed with a mixture of synthetic effluent and sewage, and the treatment efficiency was evaluated through the monitoring of the following parameters: chemical oxygen demand (COD), free cyanide, thiocyanate, copper, iron and zinc concentrations. A well adapted microbial consortium was obtained at the end of the acclimatisation period, which was able to remove more than 95% of free cyanide, thiocyanate, copper and zinc, originally found in the influent. These removal efficiencies were obtained when the reactor was operated with a hydraulic retention time (HRT) of about 8 h. The performance results of experiments carried out with lower HRT (5 h) and higher dissolved oxygen (DO) concentration (6.5 mg litre(-1)) are also presented and discussed in this study.