Extraction of chlorophylls and carotenoids from dry and wet biomass of isolated Chlorella Thermophila: Optimization of process parameters and modelling by artificial neural network

Chlorophylls and carotenoids can be extracted from microalgae using various solvents. However, there is lack of studies regarding the comparison of extraction yield of these pigments from wet and dry microalgal biomass using different combination of cell disruption methods. Therefore, in this work, we have investigated the comparison of the extraction yield of chlorophylls and carotenoids from the wet and heat-dried microalgal biomass (isolated Chlorella thermophila) using ethanol. Extraction parameters such as homogenisation time, homogenisation speed, solvent temperature, solid-solvent ratio, boiling time and microwave time have been optimised. Chlorophyll extraction yield was observed to be 2.7 fold higher from wet biomass than dry biomass while carotenoid yield was 6.7 fold higher. Highest chlorophyll yield (∼60 mg/g-dry biomass) was observed at 6 min of homogenisation time, 10,000 rpm, solid solvent ratio of 1 mg/mL and 58 °C of solvent temperature from wet biomass with extraction efficiency of ∼94 %. Highest carotenoid yield was noticed following the same conditions of chlorophyll extraction except 4 °C of solvent temperature. The modelling of the extraction process was performed using artificial neural network (ANN) which may be useful for the scale-up of the extraction process at the industrial level.     

Modeling and optimising of dextrose fermentation using a fluorosensor

Dextrose has been fermented in a Tokyo Rikakikai fermentor at five different temperatures ranging from 28 to 31v°C with Saccharomyces cerevisiae (Baker's yeast) as seeding. The progress of the reaction was recorded by measuring the fluorescence signal due to intracellular reduced nicotinamide adenine di nucleotide (NADH) present in the cells with a Dr. Ingold (Switzerland) fluorosensor which has an excitation wavelength of 360 nm and measurement wavelength of 450 nm. The optimum temperature which gave maximum growth rate and maximum biomass concentration in minimum time was found to be 28v°C. The fluorescent voltage data fitted a first order model with an average absolute deviation of less than one percent. Development of this model is useful in design of model predictive controllers.     

Production and extraction of pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger

In this work soy and wheat bran were employed as raw materials for the production of pectinases by Aspergillus niger through solid-state fermentation. Several fermentation and recovery parameters were studied. The kinetics of enzyme synthesis was investigated in the range from 13 to 96 h with moisture contents varying from 25% to 70% (w/w). A medium moisture content of 40% and a fermentation time of 22 h were selected, as these conditions resulted in high pectolytic activity and enhanced polygalacturonase productivity. In order to optimise the recovery step, the best combination of temperature of extraction, contact time and solvent type was investigated. Acetate buffer (pH 4.4), 35°C and 30 min provided the best recovery. The present results show that optimising the extraction conditions is a simple way of obtaining more concentrated enzyme extracts and could be a useful instrument to extract more selectively a desired biomolecule from fermented solids.     

Studies on the production and application of cellulase from Trichoderma reesei QM-9414

High yielding mutant strain, Trichoderma reesei QM-9414, was employed for the cellulase enzyme production. Enzyme production conditions (pH, inoculum age and concentration, and organic supplements) were optimized. The ability of partially purified enzyme to hydrolyze various regionally abundant lignocellulosic raw materials was studied. Enzymatic hydrolysis conditions (temperature, pH, enzyme and substrate concentrations) were optimized. Temperature 50v°C, pH 4.5, enzyme concentration 40 FPU/g substrate and substrate concentration 2.5% were found to be optimum for the maximum yields of sugars. #-glucosidase supplementation was found to increase both the sugar yield and hydrolysis rate, and shorten the reaction time significantly.     

响应面法优化水酶法提取辣木籽蛋白质的工艺及其功能性质研究

为了提取和利用辣木籽蛋白质,本文通过水酶法优化其提取条件,并探讨其功能性质。以辣木籽为原材料,以蛋白质提取率为指标,首先确定了最佳使用酶为Alcalase碱性蛋白酶,再通过单因素试验考察料液比、时间、温度、酶添加量和pH等因素对蛋白质提取率的影响,在此基础上,利用响应面试验设计优化水酶法提取辣木籽蛋白质的工艺。结果表明,最佳工艺条件为:使用Alcalase碱性蛋白酶,在料液比为1∶10,酶添加量为4.5%,pH为9.0,温度为60℃,时间为4.5 h,此时辣木籽蛋白质的提取率最高为68.23%。在pH为10、温度为55℃时辣木籽蛋白质的氮溶解指数最高;辣木籽蛋白质持水性随着pH的增加而增加,在温度为40℃时持水性最好;在温度为55℃时,辣木籽分离蛋白的吸油效果最明显。     

Optimization of extraction and purification of glucoamylase produced by Aspergillus awamori in solid-state fermentation

Various parameters such as solvent selection, concentration, soaking time, and temperature were tested in a single bioreactor in order to determine optimum extraction conditions of glucoamylase, when produced simultaneously with protease by Aspergillus awamari nakazawa MTCC 6652. Optimum conditions were achieved in a 10% glycerol solution soaked for 2 h at 40°C, followed by concentration of extracted glucoamylase (9,157 U/gds) by acetone precipitation (1:2, v/v), which yielded 51.9% recovery. Ion exchange chromatography and gel filtration showed specific activities of 270.5 and 337.5 U/mg, respectively, while SDS-PAGE and zymogram analysis of glucoamylase indicated the presence of three starch-hydrolyzing isoforms with molecular weights of approximately 109.6, 87.1, and 59.4 kDa, respectively     

Genotypic variation in the response of two perennial grass species to elevated carbon dioxide

Shoot and reproductive biomass of genotypes of Bromus erectus and Dactylis glomerata grown in competition at ambient and elevated CO₂ were examined for 2 consecutive years in order to test whether genetic variation in those traits exists and whether it is maintained over time. At the species level, a positive CO₂ response of shoot biomass of both species was only found in the first year of treatment. At the genotype level, no significant CO₂2genotype interaction was found at any single harvest either for vegetative or reproductive biomass of either species. Analysis over time, however, indicated that there is a potential for evolutionary adaptation only for D. glomerata: (1) repeated measures ANOVA detected a marginally significant CO₂2genotype2time interaction for shoot biomass, because the range of the genotypes CO₂ response increased over time; (2) genotypes that displayed the highest response during the first year under elevated CO₂ also showed the highest response the second year. Null (B. erectus) or weak (D. glomerata) selective potentials of elevated CO₂ were detected in this experiment, but short time series could underestimate this potential with perennial species.     

Recovery of polyhydroxybutyrate (PHB) from Cupriavidus necator biomass by solvent extraction with 1,2‐propylene carbonate

An integrated procedure for the recovery of polyhydroxybutyrate (PHB) produced by Cupriavidus necator based on the extraction with 1,2‐propylene carbonate was evaluated. The effect of temperature (100–145°C) and contact time (15–45 min), precipitation period, and biomass pretreatments (pH shock and/or thermal treatments) on PHB extraction efficiency and polymer properties was evaluated. The highest yield (95%) and purity (84%) were obtained with the combination of a temperature of 130°C and a contact time of 30 min, with a precipitation period of 48 h. Under these conditions, PHB had a molecular weight of 7.4×10⁵, which was the highest value obtained. Lower values (2.2×10⁵) were obtained for higher temperatures (145°C), while lower temperatures resulted in incomplete extraction yields (45–54%). No further yield improvement was achieved with the pH/heat pretreatments, but the polymer's molecular weight was increased to 1.3×10⁶. The PHB physical properties were not significantly affected by any of the tested procedures, as shown by the narrow ranges obtained for the glass transition temperature (4.8–5.0°C), melting temperature (170.1–180.1°C), melting enthalpy (77.8–88.5 J/g) and crystallinity (55–62%). 1,2‐Propylene carbonate was shown to be an efficient solvent for the extraction of PHB from biomass. The precipitation procedure was found to highly influence the polymer recovery and its molecular weight. Although polymer molecular weight and purity were improved by applying pH/heat pretreatment to the biomass, the procedure involves the use of large amounts of chemicals, which increases the recovery costs and makes the process environmentally unfriendly.     

Agar extraction process for Gracilaria cliftonii ()

Agar extraction process was developed for Gracilaria cliftonii by investigating the effects of various extraction variables and alkali treatments on agar yield and properties. The tested variables were soaking time, soaking temperature, seaweed to water ratio of, extraction temperatures and extraction time. Alkali treatments were carried out in alkali concentrations of 1%, 2%, 3% and 5% in a water bath at 60, 70 and 80 °C prior to agar extraction. The results showed that agar yield was significantly affected by all the tested variables. The agar yield was maximised when extraction process was carried out with 1 h soaking time at 30 °C with seaweed to water ratio of 1:150 and extracted for 3 h at 100 °C. The alkali-temperature combinations significantly influenced agar yield and properties. Irrespective of temperature, alkali treatments at 3% and 5% significantly increased the gel strength.     

Optimization of enzymatic hydrolysis of mango kernel starch by response surface methodology

Amyloglucosidase (EC 3.2.1.3) from Aspergillus niger was employed for the saccharification of mango (Mangifera indica Linn) kernel starch. Response surface methodology based on a three-level three-factor Box-Behnken design was employed to optimize the important process variables such as substrate concentration (137.5-412.5 mg), enzyme concentration (4-12 mg) and temperature (35-55 °C). The sugar yield increased with both enzyme concentration and temperature, and decreased with substrate concentration. The response surface model indicated optimum conditions (substrate, 137.5 mg; enzyme, 12 mg; temperature, 55 °C) for obtaining 0.4851 mg sugar/mg substrate, which was also verified experimentally.     

Optimizing a continuous flow lipid extraction system (CFLES) used for extracting microalgal lipids

A laboratory‐made continuous flow lipid extraction system (CFLES) was devised to extract lipids from microalgae Nannochloropsis sp., a potential feedstock for biodiesel fuel, with a focus to assess the workable temperatures and pressures for future industrial applications. Using conventional solvents, the CFLES recovered 100% of the lipids extracted with conventional Soxhlet extraction. The optimum temperature and pressure were found to be 100 °C and 50 psi, respectively; conditions significantly lower than those normally used in pressurized liquid extractions requiring specialized equipment. Approximately 87% of the extracted oil was successfully transesterified into biodiesel fuel (fatty acid methyl esters). Preliminary calculations based on the tested lab‐scale system indicated savings in energy, solvent consumption, and extraction time as 96%, 80%, and more than 90%, respectively, as compared to Soxhlet extraction. However, the true cost savings can only be assessed at scaled up level. Energy efficiency of CFLES was calculated as 48.9%. Residual water (~70%) in the biomass had no effect on the extraction performance of CFLES, which is expected to help the process economics at scaled up application. The effect of temperature and pressure on the fatty acids profile of Nannochloropsis sp. is also discussed. Based on the existing literature, the authors believe that a pressurized liquid extraction system with continuous solvent flow has not been reported for lipid extraction from Nannochloropsis sp.     

Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of enzyme leaching conditions using response surface methodology

The optimization of parameters for the effective leaching of thermostable pullulanase from Clostridium thermosulfurogenes SV2-fermented bran was carried out using response surface methodology based on the central composite rotatable design. The design contains a total of 54 experimental trials with the first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. The design was employed by selecting solvent to wheat bran ratio (S/BB), process temperature, solvent pH, shaking (RPM) and contact time (h) as model factors. Among the five independent variables studied, the S/BB, solvent pH and shaking were found to be significant. S/BB ratio of 9.0, 200 RPM shaking and solvent pH 6.0 were identified as optimum for the leaching of thermostable pullulanase from the strain SV2-fermented bran.     

In-situ biomass characterisation by impedance spectroscopy using a full-bridge circuit

A full-bridge sensor system for characterising biosuspensions by impedance spectroscopy which avoids cross-sensitivity to temperature is presented. To record a large range of different kinds of cells, frequency is varied between 500 kHz and 50 MHz. The characteristic frequencies of the #-polarisation of animal cells (mouse), yeasts (Saccharomyces cerevisiae) and Escherichia coli bacteria are located in this range. The widespread applicability of this method is demonstrated by measurements on suspensions of different cells and the determination of concentration in a fermentation process. Furthermore, yeast suspensions are characterised at temperatures between T₁=24°C and T₂=36°C to evaluate the influence of temperature. A reduction of cross-sensitivity of about one order of magnitude compared to a single-sensor results. Changing in the conductivity of the suspension by 26% alters the measured biomass concentration by only 2.8%.     

Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01

A mesophilic bacterium, Aeromonas veronii PG01, isolated from industrial wastes produced an extracellular thermostable organic solvent tolerant protease. The optimum condition for cell growth and protease production was pH 7.0 and 30 °C. The protease produced was purified 53-fold to homogeneity with overall yield of 32%, through ammonium sulphate precipitation, ion-exchange and gel permeation chromatography (GPC). The molecular weight, as determined by GPC–HPLC, was found to be about 67 kDa. SDS-PAGE revealed that the enzyme consisted of two subunits, with molecular weight of 33 kDa. The protease was active in broad range of pH from 6.0 to 10.0 with optimum activity at pH 7.5. The optimum temperature for this protease was 60 °C. The enzyme remained active after incubation at 50–60 °C for 1 h. This enzyme was stable and active after incubation with benzene and it was activated 1.3- and 1.5-fold by n-hexane and n-dodecane, respectively. This protease was inhibited completely by the classic metalloprotease inhibitor 1,10-phenanthroline and partially by the metal chelator EDTA but not by the serine protease inhibitor PMSF. The PG01 protease was found to contain 1.901 mol of zinc per mole of enzyme upon analysis by Inductively coupled plasma-optical emission spectroscopy. The thermostable and solvent tolerance property make it an attractive and promising biocatalyst for enzyme mediated synthesis.     

Microbial production of gallic acid by modified solid state fermentation

Bioconversion of tannin to gallic acid from powder of teri pod (Caesalpinia digyna) cover was achieved by the locally isolated fungus, Rhizopus oryzae, in a bioreactor with a perforated float for carrying solid substrate and induced inoculum. Modified Czapek-Dox medium, put beneath the perforated float, with 2% tannic acid at pH 4.5, temperature 32°C, 93% relative humidity, incubated for 3 days with 3-day-old inoculum was optimum for the synthesis of tannase vis-à-vis gallic acid production. Conversion of tannin to gallic acid was 90.9%. Diethyl ether was used as the solvent for extraction of gallic acid from the fermented biomass. Received 14 December 1998/ Accepted in revised form 17 June 1999     

水酶法提取紫苏籽油脂工艺

采用中心组合设计(CCD)-响应面(RSM)优化紫苏籽油脂的水酶法提取工艺。在单因素试验的基础上采用中心组合设计方法,研究了酶的种类、酶解温度、pH、液(mL)固(g)比、加酶量、以及时间相互作用对紫苏油脂提取率的影响。结果显示,拟合得到方程显著,确定的紫苏油脂提取最优条件为:碱性蛋白酶在pH9.5条件下液(mL)固(g)比9.97∶1、加酶量2.75%、温度56.1℃、时间5.25h,该条件下紫苏油脂的提取率可达到37.65%,与理论值38.3%十分接近,建立的模型真实可靠,确定了紫苏油脂的最佳提取工艺。经气相色谱检测紫苏籽油中含有棕榈酸、硬脂酸、油酸、亚油酸、α-亚麻酸等脂肪酸,水酶法提取紫苏油脂的α-亚麻酸相对含量最高67.9%,且相对溶剂法及冷榨法理化指标最好。     

Production and recovery of an alkaline exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation using statistical approach

The empirical models developed through two independent RSM (RSM-I, 2(3); RSM-II, 2(5)) in terms of effective operational factors of inoculum age, inoculum volume, wheat bran-to-moisture ratio (RSM-I) and contact time, extraction temperature, agitation, fermented bran-to-solvent ratio and SDS (RSM-II) were found adequate to describe the optimization of exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation (SSF) conditions. Through the analysis of RSM-I, wheat bran-to-moisture ratio and inoculum volume were found to be the most significant factors and an increment in both had a positive effect in enhancing enzyme yield, while in RSM-II all the factors significantly affected enzyme recovery except fermented bran-to-solvent ratio, which had the least impact within the ranges investigated in enhancing enzyme recovery. Based on contour plots and variance analysis, optimum operational conditions for maximum exo-polygalacturonase yield were achieved when 1.5% (v/w) of 24h old (OD(600 nm) approximately 2.7+/-0.2) B. subtilis RCK cells were inoculated on moistened wheat bran (1:7 solid substrate-to-moisture ratio) and enzyme was harvested by addition of solvent (1:6 fermented bran-to-solvent ratio) under shaking conditions (200 rpm) in presence of SDS (0.25% w/v) for 15 min at 35 degrees C. An over all 3.4 fold (1.7-fold RSM-I; 2.0 fold RSM-II) increase in enzyme production was attained because of optimization by RSM.     

Subcritical Water Hydrolysis of Microalgal Biomass for Protein and Pyrolytic Bio-oil Recovery

Microalgae are a promising source of protein and biofuels. This study involved the extraction of soluble proteins from raw microalgae using subcritical water hydrolysis followed by pyrolysis of the resulting spent microalgal biomass for bio-oil production. The extraction process produced solubilized protein in amounts up to 10 wt% of the dry biomass. The effects of hydrolysis temperature (150–220 °C), process time (90–180 min), and initial pH (2–12) on the chemical compositions and reactivity of the spent biomass as biofuel intermediates were investigated. It was found that when the temperature and time increased, the protein and carbohydrate fractions of the spent biomass were reduced, while their lipid fraction increased. A low initial pH led to lower protein content in the spent biomass. Compared with the raw microalgae, the spent biomass gave a higher yield of pyrolytic bio-oil that contained much less of the N-containing compounds and higher amounts of long-chain fatty acids (C₁₆) and C₁₄–C₂₀ long-chain hydrocarbons. In addition, enhanced energy recovery and a reduction in the energy consumption of the pyrolysis process were the other benefits acquired from the protein extraction. Therefore, subcritical water hydrolysis was considered to be an effective process to recover solubilized proteins, enhance the properties of the spent biomass, improve the energy balance of the subsequent pyrolysis process, and raise the quality of the bio-oil.     

Joint aerobic biodegradation of wastewater from table olive manufacturing industries and urban wastewater

Wastewater generated in the elaboration of table olives has been treated using activated sludge from a municipal wastewater plant after adequate acclimation. To avoid bactericide properties of some chemical structures present in this type of effluents, synthetic urban wastewater has been used to dilute the original wastewater. The main parameters affecting efficiency of biological processes have been studied. Thus, initial biomass concentration, temperature up to 303 K (upper working temperature limit = 313 K) and initial substrate concentration exerted a positive influence on COD degradation rate. The optimum pH was found to be around 7, experiencing a slight inhibition on cell activity at pH 4. Under the experimental conditions investigated other parameters like polyphenol content, absorbance at 254 nm and total organic carbon were also reduced to some extent. Only nitrates amount was increased after the biological process took place. A kinetic model based on Monod equation was proposed and applied to experimental results. The maximum specific growth rate was calculated by means of the aforementioned kinetic model. The value of this parameter as a function of temperature was fitted to an Arrhenius expression, w_max = 9.43 2 10¹⁰ exp(72021/RT) h^у (R in J mol^у K^у283 K < T < 303 K, pH , 7-10).     

Effect of environmental parameters on decolorization of textile wastewater using Phanerochaete chrysosporium

Effect of various parameters such as size of inoculum, temperature, carbon source on decolorization of textile wastewater by Phanerochaete chrysosporium was investigated. Textile wastewater decolorization occurred during the primary phase of growth and secondary metabolism in carbon and nitrogen limited medium, respectively. It was found that glucose concentration up to 0.3 g/l has considerable effect on decolorisation rate. Further, it was also found that the concentration of the organic nitrogen of the effluent stream was sufficient to furnish the decolorisation process. It was observed that the inoculum size in this case within 10% increased the decolorisation rate rapidly. It was found that the temperature rise from 20 to 38 °C enhanced the rate of decolorization. The optimum temperature for decolorisation was found to be about 35 °C. Effect of pH from 2-4 on decolorization was also investigated. It is concluded that using Phanerochaete chrysosporium, decolorization of the azo dye containing effluent of the textile industry was achieved to about 96% within 28 h of operation.