Optimization of Diluted Organic Acid Pretreatment on Rice Straw Using Response Surface Methodology

Lignocellolusic material is a substance that is resistant to be degraded by microorganisms or hydrolysis enzymes. To be used as materials for biofuel production, it needs pretreatment process to improve efficiency of hydrolysis. In this work, chemical pretreatments on rice straw using three diluted organic acids, including acetic acid, citric acid, oxalic acid, were optimized. Using Response Surface Methodology (RSM), the effect of three pretreatment parameters, acid concentration, treatment time, and reaction temperature, on pretreatment efficiency were statistically evaluated. The results indicated that dilute oxalic acid pretreatment led to the highest enhancement of enzymatic saccharification by commercial cellulase and yielded sugar up to 10.67 mg/ml when using 5.04% oxalic acid at 137.11 oC for 30.01 min. Compared to other acid pretreatment by acetic acid, citric acid, and hydrochloric acid, the maximum sugar yields are 7.07, 6.30, and 8.53 mg/ml, respectively. Here, it was demonstrated that organic acids can be used for pretreatment of lignocellulosic materials to enhance of hydrolysis process, which could be integrated to other applications for various biorefinery processes. 

The Investigation of Enzymatic Activity in the Soils under the Impact of Metallurgical Industrial Activity in Lori Marz, Armenia

Beta-glucosidase, chitinase, leucine-aminopeptidase, acid phosphomonoesterase and acetate-esterase enzyme activities in the soils under the impact of metallurgical industrial activity in Lori marz (district) were investigated. The results of the study showed that the activities of the investigated enzymes in the soils decreased with increasing distance from the Shamlugh copper mine, the Chochkan tailings storage facility and the ore transportation road. Statistical analysis revealed that the activities of the enzymes were positively correlated (significant) to each other according to the observation sites which indicated that enzyme activities were affected by the same anthropogenic factor. The investigations showed that the soils were polluted with heavy metals (Cu, Pb, As, Co, Ni, Zn) due to copper mining activity in this territory. The results of Pearson correlation analysis revealed a significant negative correlation between heavy metal pollution degree (Nemerow integrated pollution index) and soil enzyme activity. All of this indicated that copper mining activity in this territory causing the heavy metal pollution of the soils resulted in the inhabitation of the activities of the enzymes which are considered as biological catalysts to decompose organic materials and facilitate the cycling of nutrients.

Modified Poly(pyrrole) Film Based Biosensors for Phenol Detection

In order to detect and quantify the phenolic contents of a wastewater with biosensors, two working electrodes based on modified Poly(Pyrrole) films were fabricated. Enzyme horseradish peroxidase was used as biomolecule of the prepared electrodes. Various phenolics were tested at the biosensor. Phenol detection was realized by electrochemical reduction of quinones produced by enzymatic activity. Analytical parameters were calculated and the results were compared with each other.

The Improved Biofuel Cell for Electrical Power Generation from Wastewaters

Newly synthesized Polypropylene-g-Polyethylene glycol polymer was first time used for a compartment-less enzymatic fuel cell. Working electrodes based on Polypropylene-g-Polyethylene glycol were operated as unmediated and mediated system (with ferrocene and gold/cobalt oxide nanoparticles). Glucose oxidase and bilirubin oxidase was selected as anodic and cathodic enzyme, respectively. Glucose was used as fuel in a single-compartment and membrane-less cell. Maximum power density was obtained as 0.65 nW cm-2, 65 nW cm-2 and 23500 nW cm-2 from the unmediated, ferrocene and gold/cobalt oxide modified polymeric film, respectively. Power density was calculated to be ~16000 nW cm-2 for undiluted wastewater sample with gold/cobalt oxide nanoparticles including system.

Application of Statistical Approach for Optimizing CMCase Production by Bacillus tequilensis S28 Strain via Submerged Fermentation Using Wheat Bran as Carbon Source

Biofuels production has come forth as a future technology to combat the problem of depleting fossil fuels. Bio-based ethanol production from enzymatic lignocellulosic biomass degradation serves an efficient method and catching the eye of scientific community. High cost of the enzyme is the major obstacle in preventing the commercialization of this process. Thus main objective of the present study was to optimize composition of medium components for enhancing cellulase production by newly isolated strain of Bacillus tequilensis. Nineteen factors were taken into account using statistical Plackett-Burman Design. The significant variables influencing the cellulose production were further employed in statistical Response Surface Methodology using Central Composite Design for maximizing cellulase production. The optimum medium composition for cellulase production was: peptone (4.94 g/L), ammonium chloride (4.99 g/L), yeast extract (2.00 g/L), Tween-20 (0.53 g/L), calcium chloride (0.20 g/L) and cobalt chloride (0.60 g/L) with pH 7, agitation speed 150 rpm and 72 h incubation at 37oC. Analysis of variance (ANOVA) revealed high coefficient of determination (R2) of 0.99. Maximum cellulase productivity of 11.5 IU/ml was observed against the model predicted value of 13 IU/ml. This was found to be optimally active at 60oC and pH 5.5.

Investigations of Metals and Metal-Antibrowning Agents Effects on Polyphenol Oxidase Activity from Red Poppy Leaf

Heavy metals are one of the major groups of contaminants in the environment and many of them are toxic even at very low concentration in plants and animals. However, some metals play important roles in the biological function of many enzymes in living organisms. Metals such as zinc, iron, and cooper are important for survival and activity of enzymes in plants, however heavy metals can inhibit enzyme which is responsible for defense system of plants. Polyphenol oxidase (PPO) is a copper-containing metalloenzyme which is responsible for enzymatic browning reaction of plants. Enzymatic browning is a major problem for the handling of vegetables and fruits in food industry. It can be increased and effected with many different futures such as metals in the nature and ground. In the present work, PPO was isolated and characterized from green leaves of red poppy plant (Papaverr hoeas). Then, the effect of some known antibrowning agents which can form complexes with metals and metals were investigated on the red poppy PPO activity. The results showed that glutathione was the most potent inhibitory effect on PPO activity. Cu(II) and Fe(II) metals increased the enzyme activities however, Sn(II) had the maximum inhibitory effect and Zn(II) and Pb(II) had no significant effect on the enzyme activity. In order to reduce the effect of heavy metals, the effects of metal-antibrowning agent complexes on the PPO activity were determined. EDTA and metal complexes had no significant effect on the enzyme. L-ascorbic acid and metal complexes decreased but L-ascorbic acid-Cu(II)-complex had no effect. Glutathione–metal complexes had the best inhibitory effect on Red poppy leaf PPO activity.

Effect of Different Microbial Strains on Biological Pretreatment of Sugarcane Bagasse for Enzymatic Hydrolysis

Among agricultural residues, sugarcane bagasse is one of the most convincing raw materials for the production of bioethanol due to its availability, and low cost through enzymatic hydrolysis and yeast fermentation. A pretreatment step is needed to enhance the enzymatic step. In this study, sugarcane bagasse (SCB), one of the most abundant agricultural residues in Thailand, was pretreated biologically with various microorganisms of white-rot fungus—Phanerochaete sordid (SK 7), Cellulomonas sp. (TISTR 784), and strain A 002 (Bacillus subtilis isolated from Thai higher termites). All samples with various microbial pretreatments were further hydrolyzed enzymatically by a commercial enzyme obtained from Aspergillus niger. The results showed that the pretreatment with the white-rot fungus gave the highest glucose concentration around two-fold higher when compared with the others.

Enzymatic Synthesis of Olive-Based Ferulate Esters: Optimization by Response Surface Methodology

Ferulic acid has widespread industrial potential by virtue of its antioxidant properties. However, it is partially soluble in aqueous media, limiting their usefulness in oil-based processes in food, cosmetic, pharmaceutical, and material industry. Therefore, modification of ferulic acid should be made by producing of more lipophilic derivatives. In this study, a preliminary investigation of lipase-catalyzed trans-esterification reaction of ethyl ferulate and olive oil was investigated. The reaction was catalyzed by immobilized lipase from Candida antarctica (Novozym 435), to produce ferulate ester, a sunscreen agent. A statistical approach of Response surface methodology (RSM) was used to evaluate the interactive effects of reaction temperature (40-80°C), reaction time (4-12 hours), and amount of enzyme (0.1-0.5 g). The optimum conditions derived via RSM were reaction temperature 60°C, reaction time 2.34 hours, and amount of enzyme 0.3 g. The actual experimental yield was 59.6% ferulate ester under optimum condition, which compared well to the maximum predicted value of 58.0%.

The Effect of Enzymatic Keratin Hydrolyzate on the Susceptibility of Cellulosic-Elastomeric Material to Biodecomposition

Polymeric materials have become an integral part of every aspect of today's industry. They have wide applications, inter alia, in areas such as medicine, food industry and agriculture. In agriculture, for example, they are used for the production of pots, irrigation systems and for soil mulching. The aim of this study was the attempt to produce a biodecomposable agricultural mat, by coating cotton fabric with a blend of carboxylated styrene-butadiene latex (LBSK) containing the enzymatic hydrolyzate of keratin from cattle hair, which would serve as a material for mulching. The production of such material allows the beneficial management of burdensome tannery waste constituted by keratin from cattle hair and at the same time, the production of agricultural mats that much faster undergo decomposition than commonly used polyethylene mats.

Statistical Modeling for Permeabilization of a Novel Yeast Isolate for β-Galactosidase Activity Using Organic Solvents

The hydrolysis of lactose using β-galactosidase is one of the most promising biotechnological applications, which has wide range of potential applications in food processing industries. However, due to intracellular location of the yeast enzyme, and expensive extraction methods, the industrial applications of enzymatic hydrolysis processes are being hampered. The use of permeabilization technique can help to overcome the problems associated with enzyme extraction and purification of yeast cells and to develop the economically viable process for the utilization of whole cell biocatalysts in food industries. In the present investigation, standardization of permeabilization process of novel yeast isolate was carried out using a statistical model approach known as Response Surface Methodology (RSM) to achieve maximal b-galactosidase activity. The optimum operating conditions for permeabilization process for optimal β-galactosidase activity obtained by RSM were 1:1 ratio of toluene (25%, v/v) and ethanol (50%, v/v), 25.0 oC temperature and treatment time of 12 min, which displayed enzyme activity of 1.71 IU /mg DW.

Bioactivity Evaluation of Cucurbitin Derived Enzymatic Hydrolysates

After cold pressing of pumpkin oil, the defatted oil cake (PUOC) was utilised as raw material for processing of bio-functional hydrolysates. In this study, the in vitro bioactivity of an alcalase (AH) and a pepsin hydrolysate (PH) prepared from the major pumpkin 12S globulin (cucurbitin) are compared. The hydrolysates were produced at optimum reaction conditions (temperature, pH) for the enzymes, during 60min. The bioactivity testing included antioxidant and angiotensin I converting enzyme inhibitory activity assays. The hydrolysates showed high potential as natural antioxidants and possibly antihypertensive agents in functional food or nutraceuticals. Additionally, preliminary studies have shown that both hydrolysates could exhibit modest α-amylase inhibitory activity, which indicates on their hypoglycemic potential.

New Kinetic Approach to the Enzymatic Hydrolysis of Proteins – A Case of Thermolysin-Catalyzed Albumin

Using an enzyme of known specificity the hydrolysis of protein was carried out in a controlled manner. The aim was to obtain oligopeptides being the so-called active peptides or their direct precursors. An original way of expression of the protein hydrolysis kinetics was introduced. Peptide bonds contained in the protein were recognized as a diverse-quality substrate for hydrolysis by the applied protease. This assumption was positively verified taking as an example the hydrolysis of albumin by thermolysin. Peptide linkages for this system should be divided into at least four groups. One of them is a group of bonds non-hydrolyzable by this enzyme. These that are broken are hydrolyzed at a rate that differs even by tens of thousands of times. Designated kinetic constants were k'F = 10991.4 L/g.h, k'M = 14.83L/g.h, k'S about 10-1 L/g.h for fast, medium and slow bonds, respectively. Moreover, a procedure for unfolding of the protein, conducive to the improved susceptibility to enzymatic hydrolysis (approximately three-fold increase in the rate) was proposed.

Mathematical Modeling of Uncompetitive Inhibition of Bi-Substrate Enzymatic Reactions

Currently, mathematical and computer modeling are widely used in different biological studies to predict or assess behavior of such a complex systems as a biological are. This study deals with mathematical and computer modeling of bi-substrate enzymatic reactions, which play an important role in different biochemical pathways. The main objective of this study is to represent the results from in silico investigation of bi-substrate enzymatic reactions in the presence of uncompetitive inhibitors, as well as to describe in details the inhibition effects. Four models of uncompetitive inhibition were designed using different software packages. Particularly, uncompetitive inhibitor to the first [ES1] and the second ([ES1S2]; [FS2]) enzyme-substrate complexes have been studied. The simulation, using the same kinetic parameters for all models allowed investigating the behavior of reactions as well as determined some interesting aspects concerning influence of different cases of uncompetitive inhibition. Besides, it has been shown that uncompetitive inhibitors exhibit specific selectivity depending on mechanism of bi-substrate enzymatic reaction. 

Impact of Nonthermal Pulsed Electric Field on Bioactive Compounds and Browning Activity in Emblica officinalis Juice

The effect of nonthermal pulsed electric field (PEF) and thermal treatment (90⁰C for 60s) was studied on quality parameters of emblica officinalis juice for the period of 6 weeks at 4⁰C using monopolar rectangular pulse of 1µs width. The PEF treatment was given using static chamber at 24kV/cm for 500µs. The quality of emblica officinalis juice was investigated in terms of non enzymatic browning index (NEBI), 5-hydroxymethyl-2-furfural (HMF), total polyphenol content and antioxidant capacity. ⁰Brix, pH and conductivity were evaluated as physical parameters. The aim of the work was to investigate the effect of PEF on the retention of bioactive compounds and retardation of browning activity. The results showed that conventional thermal treatment had led to a significant (p < 0.05) decrease of 48.15% in polyphenol content (129.56 mg of GAE L-1), with higher NEBI and HMF formation (p < 0.05) whilst PEF suppressed NEBI and retained higher polyphenol compounds (168.59 mg GAE L-1) with limiting the loss to 32.56% along maximum free radical scavenging activity (92.07%). However, pH, ⁰brix and electrical conductivity of treated juice samples remain unaffected. Therefore, PEF can be considered as an effective nonthermal treatment for retaining bioactive compounds along suppressing browning of emblica juice.  

Thermal and Morphological Evaluation of Chemically Pretreated Sugarcane Bagasse

Enzymatic hydrolysis is one of the major steps involved in the conversion from sugarcane bagasse to yield ethanol. This process offers potential for yields and selectivity higher, lower energy costs and milder operating conditions than chemical processes. However, the presence of some factors such as lignin content, crystallinity degree of the cellulose, and particle sizes, limits the digestibility of the cellulose present in the lignocellulosic biomasses. Pretreatment aims to improve the access of the enzyme to the substrate. In this study sugarcane bagasse was submitted chemical pretreatment that consisted of two consecutive steps, the first with dilute sulfuric acid (1 % (v/v) H2SO4), and the second with alkaline solutions with different concentrations of NaOH (1, 2, 3 and 4 % (w/v)). Thermal Analysis (TG/ DTG and DTA) was used to evaluate hemicellulose, cellulose and lignin contents in the samples. Scanning Electron Microscopy (SEM) was used to evaluate the morphological structures of the in natura and chemically treated samples. Results showed that pretreatments were effective in chemical degradation of lignocellulosic materials of the samples, and also was possible to observe the morphological changes occurring in the biomasses after pretreatments.

Enzymatic Activity of Alfalfa in a Phenanthrene-contaminated Environment

This research was undertaken to study enzymatic activity in the shoots, roots, and rhizosphere of alfalfa (Medicago sativa L.) grown in quartz sand that was uncontaminated and contaminated with phenanthrene at concentrations of 10 and 100 mg kg-1. The higher concentration of phehanthrene had a distinct phytotoxic effect on alfalfa, inhibiting seed germination energy, plant survival, and biomass accumulation. The plant stress response to the environmental pollution was an increase in peroxidase activity. Peroxidases were the predominant enzymes in the alfalfa shoots and roots. The peroxidase profile in the shoots differed from that in the roots and had different isoenzyme numbers. 2,2'-Azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS) peroxidase was predominant in the shoots, and 2,7-diaminofluorene (2,2-DAF) peroxidase was predominant in the roots. Under the influence of phenanthrene, the activity of 2,7-DAF peroxidase increased in the shoots, and the activity of ABTS peroxidase increased in the roots. Alfalfa root peroxidases were the prevalent enzyme systems in the rhizosphere sand. Examination of the activity of alfalfa root peroxidase toward phenanthrene revealed the possibility of involvement of the plant enzyme in rhizosphere degradation of the PAH.

Enhanced Mycophenolic Acid Production by Penicillium brevicompactum with Enzymatically Hydrolyzed Casein

Mycophenolic acid (MPA) is a secondary metabolite produced by Penicillium brevicompactum, which has antibiotic and immunosuppressive properties. In this study, the first, mycophenolic acid was produced in a fermentation process by Penicillium brevicompactum MUCL 19011 in shake flask using a base medium. The maximum MPA production, product yield and productivity of process were 1.379 g/L, 18.6 mg/g glucose and 4.9 mg/L. h, respectively. Also the glucose consumption, biomass and MPA production profiles were investigated during batch cultivation. Obtained results showed that MPA production starts approximately after 180 hours and reaches to a maximum at 280 h. In the next step, the effects of some various concentrations of enzymatically hydrolyzed casein on MPA production were evaluated. Maximum MPA production, product yield and productivity as 3.63 g/L, 49 mg/g glucose and 12.96 mg/L.h, respectively were obtained with using 30 g/L enzymatically hydrolyzed casein in culture medium. These values show an enhanced MPA production, product yield and process productivity pr as 116.8%, 132.8% and 163.2%, respectively.

Statistical Optimization of the Enzymatic Saccharification of the Oil Palm Empty Fruit Bunches

A statistical optimization of the saccharification process of EFB was studied. The statistical analysis was done by applying faced centered central composite design (FCCCD) under response surface methodology (RSM). In this investigation, EFB dose, enzyme dose and saccharification period was examined, and the maximum 53.45% (w/w) yield of reducing sugar was found with 4% (w/v) of EFB, 10% (v/v) of enzyme after 120 hours of incubation. It can be calculated that the conversion rate of cellulose content of the substrate is more than 75% (w/w) which can be considered as a remarkable achievement. All the variables, linear, quadratic and interaction coefficient, were found to be highly significant, other than two coefficients, one quadratic and another interaction coefficient. The coefficient of determination (R2) is 0.9898 that confirms a satisfactory data and indicated that approximately 98.98% of the variability in the dependent variable, saccharification of EFB, could be explained by this model.

Higher Plants Ability to Assimilate Explosives

The ability of agricultural and decorative plants to absorb and detoxify TNT and RDX has been studied. All tested 8 plants, grown hydroponically, were able to absorb these explosives from water solutions: Alfalfa > Soybean > Chickpea> Chikling vetch >Ryegrass > Mung bean> China bean > Maize. Differently from TNT, RDX did not exhibit negative influence on seed germination and plant growth. Moreover, some plants, exposed to RDX containing solution were increased in their biomass by 20%. Study of the fate of absorbed [1-14ðí]-TNT revealed the label distribution in low and high-molecular mass compounds, both in roots and above ground parts of plants, prevailing in the later. Content of 14ðí in lowmolecular compounds in plant roots are much higher than in above ground parts. On the contrary, high-molecular compounds are more intensively labeled in aboveground parts of soybean. Most part (up to 70%) of metabolites of TNT, formed either by enzymatic reduction or oxidation, is found in high molecular insoluble conjugates. Activation of enzymes, responsible for reduction, oxidation and conjugation of TNT, such as nitroreductase, peroxidase, phenoloxidase and glutathione S-transferase has been demonstrated. Among these enzymes, only nitroreductase was shown to be induced in alfalfa, exposed to RDX. The increase in malate dehydrogenase activities in plants, exposed to both explosives, indicates intensification of Tricarboxylic Acid Cycle, that generates reduced equivalents of NAD(P)H, necessary for functioning of the nitroreductase. The hypothetic scheme of TNT metabolism in plants is proposed.

Biodegradation of Lignocellulosic Residues of Water Hyacinth (Eichhornia crassipes) and Response Surface Methodological Approach to Optimize Bioethanol Production Using Fermenting Yeast Pachysolen tannophilus NRRL Y-2460

The objective of this research was to investigate biodegradation of water hyacinth (Eichhornia crassipes) to produce bioethanol using dilute-acid pretreatment (1% sulfuric acid) results in high hemicellulose decomposition and using yeast (Pachysolen tannophilus) as bioethanol producing strain. A maximum ethanol yield of 1.14g/L with coefficient, 0.24g g-1; productivity, 0.015g l-1h-1 was comparable to predicted value 32.05g/L obtained by Central Composite Design (CCD). Maximum ethanol yield coefficient was comparable to those obtained through enzymatic saccharification and fermentation of acid hydrolysate using fully equipped fermentor. Although maximum ethanol concentration was low in lab scale, the improvement of lignocellulosic ethanol yield is necessary for large scale production.