Enzymatic Pretreatment Research Articles

Production of biomethane, biohydrogen, and volatile fatty acids from Nordic phytoplankton biomass grown in blended wastewater

Upgrading carbon-negative microalgal biomass to biofuels and value-added products presents a three-pronged solution for waste treatment, carbon capture, and economically viable bioenergy production. Acidogenesis and methanogenesis are versatile processes at the core of anaerobic digestion systems, facilitating the conversion of diverse biogenic substrates into energy and a wide range of biobased products. The present study was conducted to integrate acidogenesis and methanogenesis for coproduction of biohydrogen, biomethane, and volatile fatty acids from Nordic phytoplankton consortia. For this purpose, microalgal consortia was cultivated in a raceway pond equipped with high surface area structures. Harvested microalgal biomass was subjected to thermoalkaline (2% NaOH solution at 121 °C) and enzymatic (cellulase) pretreatments. The hydrolysates of the pretreated biomass were inoculated with thermally treated sludge for acidogenic fermentation and with mixture of untreated sludge and cow dung (1:1 v/v ratio) for anerobic digestion. The acidogenic process produced a significant amount of biohydrogen (maximum 164.8 mL bioH2/VSload) along with volatile fatty acids (maximum 7.9 g COD/L), while methanogenesis resulted in biomethane production of maximum 210.7 mL bioCH4/VSload, accompanied by an ammonium recovery of 1278 mg NH4+/L. These maximum yields were all achieved by enzymatic pretreatment of the biomass fraction harvested from high surface area brush head structures inserted in the raceway pond. These results have important implications for designing phytoplankton cultivation systems and upstream pathways to optimize energy production from carbon-negative phytoplankton biomass.

Comparison of yield and properties of banana juice extracted by hot water extraction method and different enzymes and preparation of wine from the extracted juices

AbstractThe yield and properties of banana juice extracted by enzymatic pretreatments using different enzymes and were compared among themselves and with hot water extraction. The treatments that gave higher yield and better properties of the juices were used for winemaking and the properties of the wines were also compared. A comparison of gas chromatography‐mass spectrometry analysis was also done between the juices and the wines. The various treatments were hot water bath extraction and different enzymes (amylase, cellulase, pectinase, and viscozyme) at two concentrations (0.05% and 0.1%). Juice yield, total soluble sugars, pH, titrable acidity, reducing sugars, and transmittance were analyzed. Best results for juice recovery (46.44 ± 0.50%), total soluble solids (14.8 ± 0.01 °Brix), and transmittance (86.93 ± 2.68%) were achieved using pectinase (0.1%), while viscozyme (0.1%) yielded best results for viscosity (1.49 ± 0.09 mPa.s), pH (5.01 ± 0.05), and acidity (0.277 ± 0.01%). The banana juices from these treatments were then used for wine preparation, and the physicochemical parameters were monitored during a 30‐day storage period. Within 20 days during fermentation, a reduction in total soluble solids from 22 °Brix to 6.1 °Brix was observed. Juice from banana pulp can be successfully extracted using the enzymes pectinase and viscozyme, and wine can be prepared from the enzymatically extracted juices.

Optimization of Bioethanol Production from <em>Ceratophyllum demersum</em> for hand sanitizers

Hands are the primary way to spread microorganisms, thus hand washing is the primary defence and an essential element of personal hygiene for infection control. Hand sanitizers that contain ethanol as their main constituent are used to kill a broad range of microbes. Bioethanol production has relied heavily on the use of first-generation feedstock. Therefore, the development and utilization of alternative feedstocks such as weed (Ceratophyllum demersum) and other non-food crops have gained more attention in recent times. This study explores the bioethanol production using C. demersum, a weedy species, for its potential use in hand sanitizer production. The substrate, C. demersum was subjected to mechanical pre-treatment and then pre-treated with varying concentrations of sulfuric acid, which was subsequently followed by enzymatic pre-treatment and allowed for fermentation using Saccharomyces cerevisiae. The results showed that a sulfuric acid concentration of 1 M resulted in a significantly higher amount of reducing sugar and alcohol yield compared to other concentrations, and this was selected for further studies. After optimization of fermentation parameters, a significantly higher alcohol yield of 2.6% was achieved using a S. cerevisiae inoculum concentration of 100 g/l and agitation at 150 rpm at 40°C for 36 hours. Subsequent optimization of fermentation media components further increased the alcohol yield to 3.7%, with the use of 6 g/l yeast extract, 6 g/l (NH4)2SO4, 5 g/l MgSO4, and 8 g/l KH2PO4. The resulting alcohol mixture was analyzed and found to contain 84.9% bioethanol. An agar well diffusion assay was conducted against bacteria and fungi. The results showed that all the bacterial and fungal strains were sensitive to the bioethanol extract as evidenced by the presence of an inhibition zone.

Influence of Enzymatic Pretreatment and Optimization on Hydro Distillation Recovery of Essential Oils from Allium cepa

Influence of Enzymatic Pretreatment and Optimization on Hydro Distillation Recovery of Essential Oils from Allium cepa

Structural property of extractable proteins and polysaccharides in wheat bran following a dual-enzymatic pretreatment and corresponding functionality

The current study applied dual-enzymatic treatment via alcalase and Bacillus velezensis hydrolase for enhancing extraction of proteins and polysaccharides from wheat bran and modifying their corresponding structure. Results indicated the aqueous extract by enzymatic pretreatment (referred as EHWB) had an increased content of soluble substance, in which 18.5 % increased for carbohydrates and 11.4 % increased for proteins in the extract compared to the aqueous extract without enzymes (labeled as AEWB). Furthermore, compositions with lower molecular weight of 130 kDa and < 21.1 kDa for polysaccharides and proteins, respectively, were found in EHWB. Interestingly, EHWB had a twice higher radicals scavenging than that of AEWB, and digestive property indicated EHWB had a greater peptides production although glucose release was lower in gastric phase. Importantly, this is the first study to reveal that gut microbiota fermentation of EHWB resulted in faster generation of short-chain fatty acids at initial fermentation stage (6 h), followed a higher generation of butyrate at final fermentation stage (24 h). This fermentation property might be associated with its presence of lower molecular weight substrates and even the changes in the molecular structure induced by the enzymes. This study highlights a novel approach for developing a value-added product from wheat bran.

Enzyme-enhanced acidogenic fermentation of waste activated sludge: Insights from sludge structure, interfaces, and functional microflora

Anaerobic fermentation is widely installed to recovery valuable resources and energy as CH4 from waste activated sludge (WAS), and its implementation in developing countries is largely restricted by the slow hydrolysis, poor efficiency, and complicate inert components therein. In this study, enzyme-enhanced fermentation was conducted to improve sludge solubilization from 283 to 7728 mg COD/L and to enhance volatile fatty acids (VFAs) yield by 58.6 % as compared to the conventional fermentation. The rapid release of organic carbon species, especially for tryptophan- and tyrosine-like compounds, to outer layer of extracellular polymeric substance (EPS) occurred to reduce the structural complexity and improve the sludge biodegradability towards VFAs production. Besides, upon enzymatic pretreatment the simultaneous exposure of hydrophilic and hydrophobic groups on sludge surfaces increased the interfacial hydrophilicity. By quantitative analysis via interfacial thermodynamics and XDLVO theory, it was confirmed that the stronger hydrophilic repulsion and energy barriers in particle interface enhanced interfacial mass transfer and reactions involved in acidogenic fermentation. Meanwhile, these effects stimulate the fermentation functional microflora and predominant microorganism, and the enrichment of the hydrolytic and acid-producing bacteria in metaphase and the proliferation of acetogenic bacteria, e.g., Rubrivivax (+9.4 %), in anaphase also benefits VFAs formation. This study is practically valuable to recovery valuable VFAs as carbon sources and platform chemicals from WAS and agriculture wastes.

Extraction and in vitro active evaluation of essential oil of Acorus tatarinowii Schott rhizome rich in β-asarone using enzymatic pretreatment and solvent-free microwave-assisted method

The essential oil from fresh rhizomes of Acorus tatarinowii Schott was obtained using the EP-SFM method (an enzymatic pretreatment and solvent-free microwave-assisted method) and characterized. The optimal conditions for essential oil extraction have been obtained experimentally through experiments on single-factor and response surface-based optimization. The highest essential oil content (1.48%, w/w) was obtained under the optimal conditions by SEM, TGA, FTIR, GC-MS and acetylcholinesterase inhibition activity analysis in vitro assay. The EP-SFM method was found to be effective in improving the extraction rate of essential oils and reducing extraction time, as indicated by the results. More importantly, this method greatly increased the active component content of β-asarone (65.87%), and the content of β-asarone was increased by 18.06% compared with the conventional steam distillation method (SD). The acetylcholinesterase inhibiting activity of EP-SFM essential oil was evaluated as well, which had the best acetylcholinesterase inhibitory activity and was closest to the positive control group.

Use of Cellulase Obtained from Solid-State Fermentation of Orange and Passion Fruit Peels as an Enzymatic Pre-treatment Step for Anaerobic Digestion

Use of Cellulase Obtained from Solid-State Fermentation of Orange and Passion Fruit Peels as an Enzymatic Pre-treatment Step for Anaerobic Digestion

Study of parameters for bioethanol production from Baltic Sea macroalgae

This study aimed to investigate the possibility of producing biofuels from Baltic Sea macroalgae. The scientific value of the article consists in the adaptation of known methods of bioethanol production from seaweeds of the Baltic Sea, selection of processing parameters and bioconversion of macroalgae biomass. Capillary sequencing using amplicon cloning identified four species of macroalgae collected on the coast of the Baltic Sea: Ulva clathrata, Ulva prolifera, Cladophohora fracta, and Ulva intenstinalis. Cellulase and xylanase enzymes, as well as microorganisms Saccharomyces cerevisiae, Lachancea thermotolerans, Pachysolen tannophilus were used to treat macroalgae biomass. Before macroalgae fermentation, acid, alkaline, and enzymatic pretreatments were performed. It was found that bioethanol can be extracted from Baltic Sea macroalgae samples using any of the processing methods considered, but alkaline pretreatment yielded the highest content of 32 %. The Baltic Sea macroalgae can be a potential source of bioethanol, which are alternatives to hydrocarbon fuels.

Potential of thermophilic bacteria isolated from cow dung-grass compost for bioethanol production using floral waste

In recent years, various efforts have been invested in producing bioethanol from lignocellulosic biomass (LCB) using thermophilic bacteria. Twelve thermophilic ethanologenic bacteria were isolated from cow dung-grass compost using an enrichment technique, and the isolate, CSD6, which produced the highest bioethanol, was studied further. CSD6 utilized both pentose and hexose sugars producing ethanol, lactic acid and acetic acid as major soluble products and was identified through 16S rRNA gene sequencing as a strain of Geobacillus stearothermophilus. The highest ethanol production was found to be 25.05 mM from 5 g/L glucose, equivalent to 55% of the theoretical ethanol yield at 55 °C and initial pH 7.5. The isolate showed average tolerance to ethanol and acetic acid concentration and initial substrate loading. The bioethanol potential of CSD6 was also studied using autoclave-treated mixed floral waste (FW) as a no-cost substrate. CSD6 produced a maximum of 8.9 mM of bioethanol with a 75.47% decrease in reducing sugars using 20 g/L FW as substrate without any enzymatic pretreatment, indicating the ability of CSD6 to produce bioethanol from easily available substrates.

Saccharification of Agricultural Wastes and Clarification of Orange Juice by Penicillium rolfsii CCMB 714 Pectinase

Pectinases are enzymes used in several industrial processes. Seven agroindustrial wastes—jackfruit seed meal (Artocarpus heterophyllus), cocoa seed peel (Theobroma cacao), cocoa husks (Theobroma cacao), passion fruit husks (Passiflora edulis), mangosteen husks (Garcinia mangostana), malt residue (Hordeum vulgare) and the peach palm waste (Bactris gasipaes Kunth.)—were evaluated to produce a crude extract containing pectinase activity by Penicillium rolfsii CCMB 714. The jackfruit seed meal was chosen as the best substrate for solid-state fermentation, which was optimized with 4 mL of water as a wetting agent for 2 days at 35 °C and with a 0.5% nitrogen source, whereby the pectinase production increased by 44% (362.09 U/g). The obtained crude extract was characterized and applied to wastes saccharification and orange juice clarification. The pectinase showed better activity at a pH of 3.0 to 5.0 and 55 °C, it stably maintained over 80% of activity at 30–50 °C for up to 60 min and 1 mM CuSO4 increased the pectinase activity by 17%. The saccharification of agroindustrial wastes (cocoa husks, mangosteen husks and passion fruit husks) resulted in 126.55 µmol/mL of reducing sugars from passion fruit husks, which represents an increase of 126% after optimization (45 °C for 22 h). For the clarification of orange juice, it was possible to reduce the absorbance of the juice by 55%. These results elucidate the potential of the low-cost pectinase solution from P. rolfsii CCMB 714 cultivated in jackfruit seed meal for both the enzymatic pretreatment of plant biomass and the application in beverage industries.

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.

Design of cellulose nanofibre-based composites with high barrier properties

Gas barrier properties are very relevant in composite materials for applications so diverse such as food packaging, electronics, or old document restoration. In the present work, four different types of cellulose nanofibres (CNFs), two types of clay minerals used individually (sepiolite) or combined (sepiolite + kaolinite), and the influence of pH, were explored in the production of composite films. Neat CNFs, only mechanically treated or prepared by enzymatic pre-treatment, gave films with good mechanical and barrier properties, but the addition of minerals led to a dramatic loss of these properties. Contrarily, the use of thin and functionalized fibrils (TEMPO-oxidised or cationized CNFs) gave composite films with good mechanical, thermal and barrier properties. Superior oxygen barrier properties (oxygen transmission rate (OTR) < 0.4 cm3 m−2 day−1) were obtained using TEMPO-oxidised CNF and 20% sepiolite, and, in general, for all the composite films containing the TEMPO CNF (OTR ≤ 1.8 cm3 m−2 day−1). The cationic CNF-based composites also showed a very good oxygen barrier (OTR ≤ 8.2 cm3 m−2 day−1). The high oxygen barrier could be explained by the compactness of the films and better entanglement of the more fibrillated nanocelluloses with the mineral particles. A decrease in the pH of the suspensions led to a decrease in the film preparation time, without a major negative impact on the composite film’s properties.

Optimization of biogas potential using kinetic models, response surface methodology, and instrumental evidence for biodegradation of tannery fleshings during anaerobic digestion.

The optimization of the batch size experiment was run for a hydraulic retention time of 45 days using proteolytic enzyme pretreatment. The highest amounts of biogas were produced in comparison to conventional BDS (25:75), which is not processed with enzymes, and there was an increase in the biogas generation of 13.9 and 18.57%. The kinetic models show the goodness of fit between 0.993 and 0.998 and the correlation coefficient's value domain was [-1, 1] from a statistical perspective. The Box-Behnken design was carried out using the response surface methodology at different levels of independent parameters to optimize the process. Different instruments were evaluated to determine the chemical structure change and the contamination of the different treatments and the raw sample of tannery fleshings was determined. Thermogravimetric analysis was conducted to determine the loss of weight on thermal degradation. The Fourier transform infrared spectrometry was carried out to determine the different functional groups, such as -OH, -CH, -NH, and C-O, present in the samples of tannery fleshings. Scanning electron microscopy and energy dispersive X-ray analysis were carried out to determine the morphological alterations in the substrate, digestate, enzyme-pretreated fleshings, and the chemical composition of samples.

Synergistic effect of enzymatic pre-treatment and amylose–lipid complex construction on the physicochemical properties of maize starch

In this study, the effects of maltogenic amylase (MAA) pre-treatment and starch–fatty acid complex construction on the physicochemical properties of maize starch (MAS) were investigated. The average chain length of MAA-modified MAS was found to decrease from 18.15 to 14.92. Moreover, MAA pre-treatment of starch induced the formation of a V-type complex. This behaviour was demonstrated by the higher diffraction intensity, enzymatic resistance and short-range ordering of the samples pre-treated with MAA compared with unmodified samples. X-ray diffraction and rheological analysis revealed that the re-crystallisation peak intensities and storage modulus of MAA–MAS–lauric acid (LA)/stearic acid (SA) complexes were lower than those of MAA–starches, MAS–LA/SA complexes and control. The rate of starch re-crystallisation was effectively decreased by the combination of MAA pre-treatment and V-type complex construction. The anti-retrogradation (long-term) characteristics of the tested samples were in the following order: MAA–MAS–LA/SA complexes > MAA–starches > MAS–LA/SA complexes > control.

Intensified extraction of anthocyanins from Berberis vulgaris L. by pulsed electric field, vacuum-cold plasma, and enzymatic pretreatments: modeling and optimization

BackgroundBerberis vulgaris L. is a valuable source of natural antioxidants, polyphenols, and anthocyanins compounds. Advanced extraction methods can increase extraction efficiency. This study investigated the efficiency of pulsed electric field, vacuum-cold plasma, and enzymatic pretreatment for anthocyanins extraction of Berberis vulgaris L.ResultsTotal polyphenols (TP), total anthocyanin (TA), and physicochemical properties of Berberis vulgaris L. were investigated. The pulsed electric field at three levels of electric intensity (3000, 5000, and 7000 V/cm) and three pulse numbers (50, 75, and 100) were applied. 7000 V/cm with the pulse number of 100 was the best condition for anthocyanin extraction (amounts of anthocyanin and polyphenol extraction were 260.28 mg/L and 462.75 mg/L, respectively). The vacuum-cold plasma was carried out at the power of 60, 70, and 80 w at different times (1, 3, and 5 min). The optimum conditions for vacuum-cold plasma were 80 w for 5 min, and anthocyanin and polyphenol amounts were 256.32 mg/L and 433.71 mg/L, respectively. The optimal conditions of enzymatic pretreatment for the maximum yield were 1.5% enzyme concentration at 60 °C (the values of extracted anthocyanin and polyphenol were 279.64 mg/L and 484.93 mg/L, respectively).ConclusionsDifferent extractionpretreatments demonstrated that the enzymatic pretreatment resulted in the highest extraction of anthocyanins and polyphenols from Berberis vulgaris L. Therefore, pectinase can act as a potential assisted extraction for the extraction process.Graphical

Ultrasound and enzyme‐pretreated extraction for the valorization of pea pod proteins

AbstractPea pod (PP) is an agricultural by‐product with 10%–13% protein content which has a potential to be converted to value‐added ingredients. In this study, different protein extraction strategies including three different solvents (water, salt, and alkaline) and two different pretreatments (ultrasound [US] and enzyme such as pectinase and Viscozyme®) before conventional alkaline extraction were employed to maximize the extraction yield. The US‐pretreated alkaline extraction achieved the highest yield (21.1%) in protein extracts under optimum conditions (30% amplitude for 5 min). PP protein concentrates showed significant differences in solubility, sodium dodecyl sulfate–polyacrylamide gel electrophoresis protein patterns, scanning electron microscopy images, and color depending on the applied extraction method. All PP residues after extraction still remained protein (5.2–8.0 g/100 g) regardless of the extraction techniques, representing the challenges in protein extraction from agricultural by‐products with a lignocellulosic matrix. The combined use of US and enzyme treatments should be further investigated for higher extraction yields.Practical ApplicationsAgricultural wastes and by‐products could be potential protein sources; however, their lignocellulosic structure could be challenging for protein extraction. In the current study, different extraction strategies including ultrasound (US) and enzyme pretreatments were performed for the extraction of proteins from pea pods which is an agro‐industrial by‐product. Results suggest that US pretreatment may have promising applications for the extraction of valuable compounds from agricultural and industrial by‐products, contributing to sustainable food processing strategies.

Alternative Eco-friendly Treatment of Hollow Cellulosic Fiber-Based Hybrid Composites for Remarkable Reactive Dyeing

Egyptian Chorisia hollow fibers have been blended with cotton waste, polyester waste, and raw bamboo at various ratios and weft counts using open-end spinning. The blended fabrics of hybrid composites were subjected to atmospheric plasma, pectinase, and lipase enzyme treatments, as well as the substitution of all hazardous chemicals, as sustainable alternatives to traditional alkaline scouring. The impacts of the treatments on the hollow-based fabrics' hydrophilicity, surface morphology, and color performance have been discussed. Pretreatment is associated with considerable weight loss (~ 12%) and a wetting time of less than 5 s which represents adequate absorbency of the composite. The enzymatic treatment contributed to looser arrangement of the fibrils which indicates an effective elimination of lignin and hemicellulose. The crystalline sections were greatly influenced by enzymatic treatment, providing effective decrease of cellulose crystallinity by about 30–40% as revealed by X-ray diffraction (XRD) measurements. Compared to conventional scouring, bio-scouring discharges significantly less effluent. The treated composite using either enzyme or plasma pretreatment method produced about at least 300% higher color yield (K/S) and 100% enhanced dye fixation (% F) than the untreated fabric. The lipase pretreatment without subsequent to atmospheric plasma treatment could be enough to achieve the highest hydrophilicity and color performance level under the experimental conditions.

Low-dosage enzymatic hydrolysis of organic municipal waste for sugar and ethanol production

AbstractEnzymatic hydrolysis (EH) of the organic fraction of municipal solid waste (OFMSW) is challenging due to its complexity and heterogeneous character. This study investigated the effects of OFMSW compositional characteristics and pretreatment techniques on the efficiency of EH. The experiments involved varying enzymatic dosages and pretreatment methods, including heat pretreatment and anaerobic incubation of raw OFMSW. Remarkably, after thermal pretreatment, a saccharification yield exceeding 50% was achieved with only 1 filter paper unit (FPU) of Cellic® Ctec3 and 0.5 glucoamylase unit (AGU) of AMG® 300 L BrewQ per g of dry OFMSW. This resulted in the recovery of 239 g of sugar per kilogram of dry OFMSW. The study revealed that the decomposition of OFMSW by enzymes is limited after reaching about 50% of the sugar recovery yield, and high concentrations of inhibitors such as organic acids, alcohols, and potentially other organics and inorganics may contribute to this limitation. These compounds, however, serve as excellent preservatives, achieving contamination control during biological conversion. Sugar-rich hydrolysates were subjected to ethanol fermentation using Saccharomyces cerevisiae, leading to the conversion of most hexose sugars into ethanol, reaching 331 g of ethanol per kilogram of dry OFMSW, and indicating that the limitation of OFMSW saccharification was not due to product inhibition. These findings highlight the need for further investigation of the saccharification process of biowaste to overcome inhibitory effects and subsequently achieve higher efficiency of the following bioprocesses.

Improving Biohydrogen Production by Dark Fermentation of Milk Processing Wastewater by Physicochemical and Enzymatic Pretreatments.

Biohydrogen is considered an alternative energy reserve. Dark fermentation is one of the important green hydrogen production techniques that utilizes organic waste as raw material. It is a promising bioconversion, easy, not expensive, and cost-effective process. Milk processing wastewater (MPWW) is an organic effluent generated in large volumes on a daily basis and disposed directly into the environment. In this research, the study of biochemical hydrogen potential (BHP) test of MPWW was evaluated and used as substrate (S). A waste sludge was used as an inoculum (I) and source of bacteria. Both substrate and inoculum were analyzed and the study was based mainly on the ratio of volatile solids (VS) of inoculum and substrate subsequently, which was noted as I/S. Different substrate pretreatments were performed: ultrasonic, thermal, chemical, and enzymatic hydrolysis. The I/S ratio impact was investigated and evaluated the hydrogen production improvement. Modified Gompertz and modified Logistic kinetic models were employed for the kinetic modeling of cumulative hydrogen production values. Results show that I/S ratio of 1/4 gVS/gVS resulted from the best hydrogen production of 59.96mL during 30days of MPWW fermentation without pretreatment. It was also shown that all the adopted pretreatments enhanced hydrogen production, whereas ultrasonic pretreatment for 5min increased the production by only 14.84%. Heat pretreatment was more efficient, where the hydrogen production increased from 60 to 162mL (170% of improvement) using heat shock at 90°C for 30min. The impact of chemical pretreatment was different from a reagent to another. Pretreatment using calcium hydroxide resulted in the biggest hydrogen production of 165.3mL (175.5%) compared to the other chemical pretreatments. However, the best hydrogen production was given by the biological pretreatment using enzymatic hydrolysis (Lactase) resulting in 254mL of hydrogen production, which is equivalent to 323.62% of production improvement. Modified Gompertz and Logistic kinetic models fitted well with experimental data. Thus, the enzymatic hydrolysis of MPWW proved to be a promising technique for biohydrogen production enhancement.