Effect Of Enzymatic Pretreatment Research Articles

Enzymatic pretreatment for cellulose nanofiber production: Understanding morphological changes and predicting reducing sugar concentration

Enzymatic pretreatment plays a crucial role in producing cellulose nanofibers (CNFs) before fibrillation. While previous studies have explored how treatment severity affects CNF characteristics, there remains a lack of suitable parameters to monitor real-time enzymatic processes and fully comprehend the link between enzymatic action, fibrillation, and CNF properties. This study focuses on evaluating the impact of enzyme charge (using a monocomponent endoglucanase) and treatment time on cellulose fiber morphology and reducing sugar generation. For the first time, a random forest (RF) model is developed to predict reducing sugar concentration based on easily measurable process conditions (e.g., stirrer power consumption) and fiber/suspension characteristics like fines content and apparent viscosity. Polarized light optical microscopy was found to be a suitable technique to evaluate the morphological changes that fibers experience during enzymatic pretreatment. The research also revealed that endoglucanases initially induce surface fibrillation, releasing fine fibers into the suspension, followed by fiber swelling and shortening. Furthermore, the effect of enzymatic pretreatment on resulting CNF characteristics was studied at two fibrillation intensities, indicating that a high enzyme charge and short treatment times (e.g., 90 min) are sufficient to produce CNFs with a nanofibrillation yield of 19–23 % and a cationic demand ranging from 220 to 275 μeq/g. This work introduces a well-modeled enzymatic pretreatment process, unlocking its potential and reducing uncertainties for future upscaling endeavors.

Effect of Enzymatic Pre-Treatment on Oat Flakes Protein Recovery and Properties

Oats are considered an exceptional source of high-quality protein. Protein isolation methods define their nutritional value and further applicability in food systems. The aim of this study was to recover the oat protein using a wet-fractioning method and investigate the protein functional properties and nutritional values among the processing streams. The oat protein was concentrated through enzymatic extraction, eliminating starch and non-starch polysaccharides (NSP), treating oat flakes with hydrolases, and reaching protein concentrations of up to about 86% in dry matter. The increased ionic strength from adding sodium chloride (NaCl) improved protein aggregation and resulted in increased protein recovery. Ionic changes improved protein recovery in provided methods by up to 24.8 % by weight. Amino acid (AA) profiles were determined in the obtained samples, and protein quality was compared with the required pattern of indispensable amino acids. Furthermore, functional properties of the oat protein, such as solubility, foamability, and liquid holding capacity, were investigated. The solubility of the oat protein was below 7 %; foamability averaged below 8%. The water and oil-holding reached a ratio of up to 3.0 and 2.1 for water and oil, respectively. Our findings suggest that oat protein could be a potential ingredient for food industries requiring a protein of high purity and nutritional value.

Effect of enzymatic pre-treatment on thermophilic composting of shrimp pond sludge to improve ammonia recovery

In recent years, attempts have been made to develop a thermophilic composting process for organic sludge to produce ammonia gas for high value-added algal production. However, the hydrolysis of non-dissolved organic nitrogen in sludge is a bottleneck for ammonia conversion. The aim of this study was to identify enzymes that enhance sludge hydrolysis in a thermophilic composting system for ammonia recovery from shrimp pond sludge. This was achieved by screening useful enzymes to degrade non-dissolved nitrogen and subsequently investigating their effectiveness in lab-scale composting systems. Among the four hydrolytic enzyme classes assessed (lysozyme, protease, phospholipase, and collagenase), proteases from Streptomyces griseus were the most effective at hydrolysing non-dissolved nitrogen in the sludge. After composting sludge pre-treated with proteases, the final amount of non-dissolved nitrogen was 46.2% of the total N in the control sample and 22.3% of the total N in the protease sample, thus increasing the ammonia (gaseous and in-compost) conversion efficiency from 41.5% to 56.4% of the total N. The decrease in non-dissolved nitrogen was greater in the protease sample than in the control sample during the pre-treatment period, and no difference was observed during the subsequent composting period. These results suggest that Streptomyces proteases hydrolyse the organic nitrogen fraction, which cannot be degraded by the bacterial community in the compost. Functional potential analysis of the bacterial community using PICRUSt2 suggested that 4 (EC:3.4.21.80, EC:3.4.21.81, EC:3.4.21.82, and EC:3.4.24.77) out of 13 endopeptidase genes in S. griseus were largely absent in the compost bacterial community and that they play a key role in the hydrolysis of non-dissolved nitrogen. This is the first study to identify the enzymes that enhance the hydrolysis of shrimp pond sludge and to show that the thermophilic bacterial community involved in composting has a low ability to secrete these enzymes.

Performance evaluation of biocatalytic and biostimulation approaches for the remediation of trace organic contaminants in municipal biosolids

Trace organic contaminants (TrOCs) in biosolids is creating potential threats for reuse of biosolids. Out of the tested 64 trace organic contaminants, seven pharmaceutically active compounds (PhACs), and three pesticides were detected in biosolids from a municipal wastewater treatment plant. This study encompasses the removal of TrOCs and improvement in the aerobic digestion of biosolids by various pretreatments including utilization of indigenous microbes present in biosolids (T1), the effect of an enzymatic pretreatment (T2), biostimulation by the addition of an external carbon source (T3) and the synergic effect of biostimulation and enzymatic pretreatment (T4). After 28 days of aerobic digestion, total PhACs removal was 44% with T1, which improved to 51%, 54% and 62% in T2, T3 and T4, respectively. Also, total pesticides removal was 10% in T1, which enhanced to 44%, 14% and 54% in T2, T3 and T4, respectively. The extracellular enzyme activities were also monitored in all the treatments and the maximum activities (114 ± 11 U/L lipase, 382 ± 29 U/L phosphatase, 155 ± 8 U/L protease, 304 ± 26 U/L amylase, 108 ± 7 U/L laccase, and 63 ± 2 U/L lignin peroxidase) were observed in T4 after 28 days of digestion. Thus, this study aids in providing changing aspects of enzyme profiles during these processes and the enhanced bioremediation of biosolids through the hydrolytic and oxidoreductase enzymes produced by the indigenous microorganisms.

Enzyme-Assisted Mechanical Peeling of Cassava Tubers

In this study, the effect of enzymatic pre-treatment and the size of cassava tubers on mechanical peeling was examined. Cassava tubers were sorted based on their mass as small, medium and large. Viscozyme® L and an abrasive cassava peeling machine was used for the enzymatic pre-treatment and the mechanical peeling, respectively. Response surface methodology (RSM) was used to investigate the effect of the enzyme dose (0.5–1.9 mL g−1), incubation time (1.5–6 h), peeling time (1.5–4.5 min) and size of the tubers (small, medium and large) on the peeling process. Peeled surface area (PSA) and peel loss (PL) were measured as main responses in RSM. Results showed that the PSA and PL were significantly (p < 0.05) influenced by the enzyme dose, incubation time and peeling time. The size of tubers only had a significant impact on the PSA. The optimum operating conditions for different sizes of tubers were found and validated. Under optimum conditions, the PSA of the large tubers (89.52%) was significantly higher than the PSA of the medium and small tubers (p < 0.05). Application of enzymatic pre-treatment can improve the mechanical peeling process especially for larger cassava tubers.

Fabricating cellulose nanofibril from licorice residues and its cellulose composite incorporated with natural nanoparticles

It is important to make full utilization of industrial biomass residues. Pulp was prepared from licorice residues by soda-anthraquinone pulping followed by peroxyacetic acid bleaching. Cellulose nanofibril was obtained by enzymatic pretreatment followed by homogenization of the pulp (ETCNF). The effects of enzymatic pretreatment on ETCNF were investigated. Chitosan nanofibril (CHN) and lignin nanoparticles (LNPs) were prepared and used for ETCNF composites, respectively. The results showed that ETCNF exhibited clear nanofibrillar structure and a highly relative colloidal stability, and a much higher crystallinity index and thermal stability compared to TEMPO-medicated oxidized one; the cellulose composite films incorporated with CHN or LNPs exhibited good thermal stability and hydrophobicity. Compared with ETCNF film, ETCNF@LNPs-5.0% film showed higher UV-blocking ability and thermal stability, but reduced light transmittance, while ETCNF@CHN-5.0% film showed improved mechanical properties and similar light transmittance. This study would expend licorice residues as potential materials for CNF and its applications.

Effect of Enzymatic Pre-treatments on Hemp Woven Fabrics

Kenevir lifi tarih boyunca tekstil materyallerinde doğal elyaf olarak kullanılmış, zaman içinde önemini kaybetmiştir. Ancak son yıllarda, tekstilde biyobozunurluğun ve sürdürülebilirliğin önem kazanması ile alternatif doğal liflerin önemi de oldukça artmıştır. Kenevir bitkisinin kontrollü ekiminin serbest bırakılması ile bu lifin tekstilde kullanım olanakları yeniden araştırılmaya başlanmıştır. Bu çalışmada, kenevir dokuma kumaşın enzimlerle ön işlem olanakları araştırılmıştır. NaOH, H2O2 ve α-amilaz, pektinaz, lakkaz enzimleri uygulanarak üç farklı yönteme göre ön terbiye sonrası kumaş performans özellikleri karşılaştırılmıştır. Yöntem 1’de amilaz ile haşılı sökülen kumaş, NaOH ile hidrofilleştirilip ardından H2O2 ile ağartılmıştır. Yöntem 2’de haşılı sökülen kumaş, pektinazla hidrofilleştirilip ardından H2O2 ile ağartılmıştır. Yöntem 3’de kumaş, Yöntem 2’ye göre hidrofilleştirilip ardından lakkazla işleme tabi tutulmuştur. Kumaşların hidrofiliteleri, renk koordinatları, sarılık indeksleri, mukavemetleri, uzamaları ve buruşmazlık açıları değerlendirilmiştir. En iyi hidrofilite (1,46 s) Yöntem 2 sonrası elde edilmiştir. Enzimatik işlemler sonrası, renklendirme öncesi uygun kumaş zemin renginin elde edilebilmesi için optimizasyon gerekliliği ortaya çıkmıştır. İşlemler kumaşların kopma mukavemetinde ve uzamasında önemli bir değişim meydana getirmemiştir. Yöntem 1’e göre işlem gören kumaşların en yüksek buruşmazlık açısına (78°) sahip olduğu görülmüştür. Sonuç olarak kenevir elyafın yakın gelecekte öneminin tekrar artacağı ve endüstriyel tekstil uygulamalarında kullanım potansiyelinin olduğu söylenebilir.

The Effect of Enzymatic Pre-Treatment in Agarwood Oil Extraction

Agarwood oil was extracted from a pre-treated agarwood chip soaked in a specific enzyme concentration of 1 %, 3 % and 5 % respectively. The effect of soaking time was also investigated by applying soaking period in the range of 3 to 14 days. Hydrodistillation was carried out using the Clevenger-type apparatus to obtain the essential oil of agarwood chips. Subsequently the quality of the oil and the identification of its oil compounds were analyzed using GC-MS. The morphology of agarwood chips prior to hydrodistillation was characterized using scanning electron microscope (SEM). The SEM results of enzymatic pre-treated sample showed an obvious change in the cell morphology as compared to the sample with standard soaking of 6 days. The highest yield of oil with the amount of 0.125 % is produced from the sample pre-treated with 3 % enzyme concentration as compared to 0.065 % of oil yield from the non-pre-treated sample employing similar soaking days. The GC-MS analysis revealed a total of 335 compounds from all samples of agarwood oil and similar compounds were found in the standard soaking and enzymatic pre-treated samples. Caryophyllene, Gurjunene, and Alloaromadendrene that contributes to the unique odour of agarwood oil are found mostly in all samples. Thus, the enzymatic pre-treatment indicates a comparable yield and oil quality and it is achievable even with a reduced soaking time of 6 days as compared to standard 14 days.

Production and purification of xylanase from alkaliphilic Bacillus licheniformis and its pretreatment of eucalyptus kraft pulp

Abstract Xylanases from bacterial sources have potential applications in various industrial processes. In the present study, the effect of enzymatic pre-treatment on pulp bleaching has been investigated using purified xylanase from alkaliphilic Bacillus licheniformis strain Alk-1. The xylanase produced by present isolate while growing on wheat bran was purified to homogeneity by 5.84-fold purification with ~15.97% recovery by anion exchange chromatography using DEAE-cellulose. The estimated molecular weight of the xylanase was ~46 kDa. The optimal pH and temperature for xylanase activity were pH 9.0 and 60 °C, respectively. The enzyme showed good activity retention (80%) after 1 h incubation at 60 °C and pH 9.0. Enzyme activity was stimulated by Ca2+, Fe2+ and Mg2+ and inhibited by Cd2+, Hg2+ and Cu2+ at 2 mM and 10 mM conc. For xylan, the enzyme gave a Km value of 5.26 mg/mL and Vmax value of 433 μM/min/mg proteins when the reaction was carried out at 60 °Cand pH 9.0. Pre-treatment of kraft pulp with xylanase showed 19% reduction in kappa number, compared to control pulp, after 2 h treatment. The SEM and FTIR analysis of xylanase-treated pulp revealed significant morphological and structural changes on pulp fibres. GC-MS analysis of filtrates from control and xylanase-treated pulps showed variation in the presence of different plant-derived organic compounds. The present work finds potential application in paper production by making it cleaner and ecofriendly process.

Palmaria palmata as an alternative protein source: enzymatic protein extraction, amino acid composition, and nitrogen-to-protein conversion factor

The red seaweed Palmaria palmata has previously been reported to have high protein content high in essential amino acids. To extract the proteins a rigid cell wall consisting mainly of β-(1→4)/β-(1→3)-D-xylans must be disrupted. Different methods have been used to overcome this problem along with various methods used for protein evaluation. In this study, the effect of enzymatic pre-treatment on protein extraction was examined. Both enzymatic hydrolysis with xylanase and protease were tested. The amino acid content of the fractions was examined after extraction. The amino acid composition was similar to what has previously been reported; P. palmata was high in essential amino acids. Accordingly, a nitrogen-to-protein conversion factor was calculated for each fraction individually and protein results were compared with calculation using the proximate 6.25 conversion factor. The nitrogen-to-protein conversion factor varied between fractions but all factors were significantly lower than the popularly used 6.25 indicating that this conversion factor for processed P. palmata is effectively and considerably overestimating the protein content. Enzymatic pre-treatment with xylanase resulted in enhanced amino acid content and successful protein extraction. Enzymatic hydrolysis using protease resulted in higher protein content in the liquid extract compared to hydrolysis with xylanase, due to the release of proteins, peptides, and amino acids. Therefore, hydrolysis with protease is not suitable to extract proteins from P. palmata with the method described within this study but might be an optimal method to examine the bioactivity by extracting the protein hydrolysates. However, the result from this study confirm that hydrolysis with xylanase is a feasible choice to extract proteins of good quality from P. palmata.

The Influence of Microbial Consortium and C/N Ratio to Biogas Production from Rice Husk Waste by Using Solid State Anaerobic Digestion (SS-AD)

Rice husk is one of agricultural waste which have a potential to be processed into biogas. The aim of this research was to study the effect of enzymatic pretreatment and C/N ratio to biogas production from rice husk by solid state anaerobic digestion (SS-AD). This research were operated in batch system and at room temperature. TS was set 21%. Enzymatic pretreatment was conducted using lignase enzyme. C/N ratio was varied from 35, 40, 45, and 50. The variation of C/N ratio is made by adding technical urea to the substrate. Biogas formed was measured by using water displacement method every two days. The result showed that enzymatic pretreatment could increase biogas production varied from 30 to 55 %. The highest biogas production was obtained at C/N ratio 35. Specific biogas production on C/N ratio of 35, 40, 45, and 50 were 11.6, 10.2, 9.8 and 9.4 ml/grTS, respectively. SS-AD has volumetric loading of biogas production higher than generally in liquid anaerobic digestion (L-AD). Combined with our previous research with variations of C/N ratio 20, 25, 30, and 35, we obtained an optimum C/N ratio at 35.

Enzymatic mitigation of acrylamide in fried potato chips using asparaginase from Aspergillus terreus

SummaryAcrylamide in foods is declared as carcinogen. In the present work, the effect of enzymatic pretreatment and other parameters like enzyme concentration, frying conditions with respect to temperature and time, size of potato chips, and effect of sodium chloride and citric acid on mitigation of acrylamide were studied. The concentration of acrylamide in fried potatoes after the pretreatment was found to be 815.63 μg kg−1. The optimised asparaginase concentration for the mitigation of acrylamide was calibrated as 4 U mL−1, and optimised frying time and temperature were 15 min and 170 °C, respectively. An in‐depth kinetic relationship for the effect of asparaginase on the mitigation of acrylamide was studied. The prime novelty of the project is focused on the immobilisation of asparaginase to nanomagnetic particles for redundant usage with stabilised enzyme activity. The work projected three stables cycles of asparaginase activity and on further usage of the immobilised enzyme resulted in decreased activity. The repeated use of immobilised asparaginase provides the advantage of decreasing cost in processing.

The effects of enzymatic pre-treatment and type of yeast on chemical properties of white wine

The study investigated 15 variants of wine differing in their must pre-treatment (maceration by enzymes: Siha Pectinase or SihazymUni) and fermentation by different yeast strains (S.cerevisiae as ICV - D-47; SIHA- Cryaroma; Challenge Aroma White; and S. bayanus as SIHA Active Yeast). The effects of experimental conditions on chemical composition (pH, acidity, sugar), content of phenolic (LC–PDA-QTOF/MS) and volatile compounds (GC-MS), antioxidant activity (ABTS and FRAP assay), color property (CIEL*a*b*), and ethanol content were measured. For all production processes, significant changes in basic parameters and in the amount of polyphenolic compounds and antioxidant activity in wine were observed as compared to must. The wines obtained with S. cerevisiae were characterized by higher polyphenol content, smaller reduction of antioxidant activity and the strongest aroma. Challenge Aroma White yeast strain particularly contributed to good quality of wine. Enzymatic pre-treatment did not significantly affect the studied parameters (expect pH and the content of phenolic acids).

Wpływ wstępnej obróbki enzymatycznej nasion na parametry procesu tłoczenia i cechy jakościowe oleju lnianego

Wpływ wstępnej obróbki enzymatycznej nasion na parametry procesu tłoczenia i cechy jakościowe oleju lnianego

Effect of enzymatic pretreatment on the extraction yield and physicochemical properties of sago starch

The aim of this research was to investigate the extraction yield and physicochemical properties of sago starch extracted after enzymatic pretreatment. Sago pith was enzymatic‐pretreated by varying enzyme (cellulase enzyme complex) concentration, pretreatment time, and sago pith slurry concentration. The extracted sago starch was analyzed for its properties, e.g., extraction yield, pasting and thermal properties, and in vitro starch digestibility. Sago pith contained 82% (db) starch. The extraction yield and reducing sugar released increased with enzyme concentration and pretreatment time. The optimum enzymatic pretreatment conditions were 10% (w/w) sago pith slurry, enzyme concentration of 0.25 mL (618 carboxymethylcellulose unit and 139 p‐nitrophenyl‐β‐d‐glucopyranoside unit)/g substrate, and 24 h pretreatment time, providing the highest extraction yield of 71.36% (w/w sago pith, db), while control method, without enzymatic pretreatment, provided 61.01% extraction yield. Results from pasting and thermal properties confirmed that the granular stability, resistance to shear, and crystalline perfection of sago starch were improved due to hydrothermal effect of annealing during enzymatic pretreatment. The enzymatic‐pretreated sago starch also had lower retrogradation but higher starch digestibility than the control sago starch. The enzymatic pretreatment enhanced the extraction yield of sago starch and also improved its quality by the effect of annealing.

Effect of enzymatic pretreatment on the anaerobic digestion of milk fat for biogas production

The lipids present in dairy wastes, in addition to representing an important industrial loss, interfere negatively in wastewater systems. Nevertheless, if properly and separately considered, this material may be an interesting substrate for methane production. The objective of the present research was to evaluate the anaerobic degradation of milk fat in natura and when separately hydrolyzed by two lipases, one produced by Geotrichum candidum (GCL) and the other produced by Candida rugosa (CRL). The main purpose was to evaluate whether the enzymes' mechanisms of action would interfere with the anaerobic digestion of fats. The rates of biogas production and specific methane production both indicated CRL as the most advantageous. In addition to offering no benefit, pre-hydrolysis with GCL showed a higher degree of microbial inhibition.

Effect of enzymatic pretreatment on anaerobic co-digestion of sugar beet pulp silage and vinasse

Results of sugar beet pulp silage (SBPS) and vinasse (mixed in weight ratios of 3:1, 1:1 and 1:3, respectively) co-fermentation, obtained in this study, provide evidence that addition of too high amount of vinasse into the SBPS decreases biogas yields. The highest biogas productivity (598.1mL/g VS) was achieved at the SBPS–vinasse ratio of 3:1 (w/w). Biogas yields from separately fermented SBPS and vinasse were by 13% and 28.6% lower, respectively. It was found that enzymatic pretreatment of SBPS before methane fermentation that caused partial degradation of component polysaccharides, considerably increased biogas production. The highest biogas yield (765.5mL/g VS) was obtained from enzymatic digests of SBPS–vinasse (3:1) blend (27.9% more than from fermentation of the counterpart blend, which was not treated with enzymes). The simulation of potential biogas production from all the aforementioned mixtures using the Gompertz equation showed fair fit to the experimental results.

Effect of enzymatic pretreatment on the mechanical properties of jute fiber-reinforced polyester composites

The effect of various enzyme treatments on the mechanical properties of jute fiber-reinforced polyester composites was investigated in this study. Prior to composite production, jute fabrics were treated with pectinase, laccase, cellulase and xylanase enzyme solutions with varying enzyme mixtures and treatment time according to experimental design. The comparison of NaOH and enzyme-treated samples was also investigated. Jute fabric-reinforced polyester composite samples were produced using compression molding. The effect of enzymatic and NaOH treatment on fibers and fiber-matrix interface was investigated by scanning electron microscopy study. The mechanical properties of enzyme-treated and control (without enzyme treatment) samples were evaluated by means of tensile and flexural tests. It was observed that enzymes destroyed pectin, hemicelluloses and lignin substances from the fiber bundle interface which reduced the technical fiber diameter and hence increased the fiber aspect ratio. Therefore, a greater fiber-matrix interface area was created after enzymatic treatment, which facilitated better fiber-matrix adhesion and improved mechanical properties of the composites. The data indicated that enzyme treatment can be used as an effective, cheap and environmentally friendly fiber modification method for natural fiber-reinforced composite production.

Effect of enzymatic pretreatment on the synthesis and properties of phosphorylated amphoteric starch

► Phosphorylated amphoteric starch was prepared from enzyme-modified starch. ► The optimum reaction parameters were studied using response surface methodology. ► Enzymatic pretreatment improved the reaction processes significantly. ► The pasting stability of amphoteric starch from enzyme-pretreated tapioca starch was improved. Phosphorylated amphoteric starch was prepared from enzyme-modified tapioca starch in the aqueous alcoholic–alkaline medium. Optimization of the reaction parameters for maximum degree of substitution (DS) of phosphorylated amphoteric starch were carried out using response surface methodology (RSM). Optimum modification conditions were shown as following: NaOH concentration 3.71 g/100 g starch (dry basis), CHPTAC concentration 6.34 mL/100 g starch (dry basis), STP concentration 6.13 g/100 g starch (dry basis), water/ethanol ratio 1.16, reaction temperature 50 °C, reaction time 3 h, and the starch concentration 30%. Under the optimal condition, the maximum anion degree of substitution (DS A ) and cation degree of substitution (DS C ) were 0.0274 and 0.0408, respectively. Compared with the DS of the amphoteric starch prepared from native tapioca starch (NPTAS), the DS of the amphoteric starch from enzyme-pretreated tapioca starch (EPTAS) was higher at the same reaction conditions, suggesting enzymatic pretreatment improved the reaction processes significantly. Scanning electron microscopy and X-ray diffraction data showed that the granular shape and crystalline structure of EPTAS did not change. Compared with NPTAS, the pasting stability of EPTAS with the same DS was also significantly improved.

Effect of enzymatic pretreatment and increasing the organic loading rate of lipid-rich wastewater treated in a hybrid UASB reactor

This study aimed at evaluating the effect of increasing organic loading rates and of enzyme pretreatment on the stability and efficiency of a hybrid upflow anaerobic sludge blanket reactor (UASBh) treating dairy effluent. The UASBh was submitted to the following average organic loading rates (OLR) 0.98 Kg.m −3.d −1, 4.58 Kg.m −3.d −1, 8.89 Kg.m −3.d −1 and 15.73 Kg.m −3.d −1, and with the higher value, the reactor was fed with effluent with and without an enzymatic pretreatment to hydrolyze fats. The hydraulic detention time was 24 h, and the temperature was 30 ± 2 °C. The reactor was equipped with a superior foam bed and showed good efficiency and stability until an OLR of 8.89 Kg.m −3.d −1. The foam bed was efficient for solid retention and residual volatile acid concentration consumption. The enzymatic pretreatment did not contribute to the process stability, propitiating loss in both biomass and system efficiency. Specific methanogenic activity tests indicated the presence of inhibition after the sludge had been submitted to the pretreated effluent. It was concluded that continuous exposure to the hydrolysis products or to the enzyme caused a dramatic drop in the efficiency and stability of the process, and the single exposure of the biomass to this condition did not inhibit methane formation.