Scholarly

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

Year: 2015 Volume: 9 Issue: 5 503 - 507 Pages

Abstract: 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.

Keywords:
Lignocellolusic biomass
pretreatment
organic acid response surface methodology
biorefinery.

Cell Biomass and Lipid Productivities of Meyerella planktonica under Autotrophic and Heterotrophic Growth Conditions

Year: 2015 Volume: 9 Issue: 5 475 - 479 Pages

Abstract: Microalgae Meyerella planktonica is a potential biofuel source because it can grow in bulk in either autotrophic or heterotrophic condition. However, the quantitative growth of this algal type is still low as it tends to precipitates on the bottom. Besides, the lipid concentration is still low when grown in autotrophic condition. In contrast, heterotrophic condition can enhance the lipid concentration. The combination of autotrophic condition and agitation treatment was conducted to increase the density of the culture. On the other hand, a heterotrophic condition was set up to raise the lipid production. A two-stage experiment was applied to increase the density at the first step and to increase the lipid concentration in the next step. The autotrophic condition resulted higher density but lower lipid concentration compared to heterotrophic one. The agitation treatment produced higher density in both autotrophic and heterotrophic conditions. The two-stage experiment managed to enhance the density during the autotrophic stage and the lipid concentration during the heterotrophic stage. The highest yield was performed by using 0.4% v/v glycerol as a carbon source (2.9±0.016 x 10^6 cells w/w) attained 7 days after the heterotrophic stage began. The lipid concentration was stable starting from day 7.

Atmospheric Fluid Bed Gasification of Different Biomass Fuels

Year: 2015 Volume: 9 Issue: 4 337 - 340 Pages

Abstract: This paper shortly describes various types of biomass and a growing number of facilities utilizing the biomass in the Czech Republic. The considerable part of this paper deals with energy parameters of the most frequently used types of biomass and results of their gasification testing. Sixteen most used "Czech" woody plants and grasses were selected; raw, element and biochemical analyses were performed and basic calorimetric values, ash composition, and ash characteristic temperatures were identified. Later, each biofuel was tested in a fluidized bed gasifier. The essential part of this paper provides results of the gasification of selected biomass types. Operating conditions are described in detail with a focus on individual fuels properties. Gas composition and impurities content are also identified. In terms of operating conditions and gas quality, the essential difference occurred mainly between woody plants and grasses. The woody plants were evaluated as more suitable fuels for fluidized bed gasifiers. Testing results significantly help with a decision-making process regarding suitability of energy plants for growing and with a selection of optimal biomass-treatment technology.

The Improved Biofuel Cell for Electrical Power Generation from Wastewaters

Year: 2015 Volume: 9 Issue: 3 292 - 295 Pages

Abstract: 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

Year: 2015 Volume: 9 Issue: 1 76 - 86 Pages

Abstract: 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.

Biohydrogen Production from Starch Residues

Year: 2014 Volume: 8 Issue: 12 1400 - 1406 Pages

Authors:
Francielo Vendruscolo

Abstract: This review summarizes the potential of starch agroindustrial residues as substrate for biohydrogen production. Types of potential starch agroindustrial residues, recent developments and bio-processing conditions for biohydrogen production will be discussed. Biohydrogen is a clean energy source with great potential to be an alternative fuel, because it releases energy explosively in heat engines or generates electricity in fuel cells producing water as only by-product. Anaerobic hydrogen fermentation or dark fermentation seems to be more favorable, since hydrogen is yielded at high rates and various organic waste enriched with carbohydrates as substrate result in low cost for hydrogen production. Abundant biomass from various industries could be source for biohydrogen production where combination of waste treatment and energy production would be an advantage. Carbohydrate-rich nitrogendeficient solid wastes such as starch residues can be used for hydrogen production by using suitable bioprocess technologies. Alternatively, converting biomass into gaseous fuels, such as biohydrogen is possibly the most efficient way to use these agroindustrial residues.

Production of 3-Methyl-1-Butanol by Yeast Wild Strain

Year: 2014 Volume: 8 Issue: 4 412 - 415 Pages

Abstract: The biomass-based fuels have become great concern in order to replace the petroleum-based fuels. Biofuels are a wide range of fuels referred to liquid, gas and solid fuels produced from biomass. Recently, higher chain alcohols such as 3-methyl-1-butanol and isobutanol have become a better candidate compared to bioethanol in order to replace gasoline as transportation fuel. Therefore, in this study, 3-methyl-1-butanol was produced through a fermentation process by yeast. Several types of yeast involved in this research including Saccharomyces cerevisiae, Kluyveromyces lactis GG799 and Pichia pastoris (KM71H, GS115 and X33). The result obtained showed that K. lactis GG799 gave the highest concentration of 3-methyl-1-butanol at 274 mg/l followed by S. cerevisiae, P. pastoris GS115, P. pastoris KM71H and P. pastoris X33 at 265 mg/l, 190 mg/l, 182 mg/l and 174 mg/l respectively. Based on the result, it proved that yeast have a potential in producing 3-methyl-1-butanol naturally.

Performance, Emission and Combustion Characteristics of a Variable Compression Ratio Diesel Engine Fueled with Karanj Biodiesel and Its Blends

Year: 2014 Volume: 8 Issue: 4 806 - 813 Pages

Abstract: The use of biodiesel in conventional diesel engines results in substantial reduction of unburned hydrocarbon, carbon monoxide and particulate matters. The performance, emission and combustion characteristics of a single cylinder four stroke variable compression ratio engine when fueled with Karanja (Pongamia) methyl ester and its 10-50 % blends with diesel (on a volume basis) are investigated and compared with standard diesel. The suitability of karanja methyl ester as a biofuel has been established in this study. The useful brake power obtained is similar to diesel fuel for all loads. Experiment has been conducted at a fixed engine speed of 1500 rpm, variable load and at compression ratios of 17.5:1 and 18.5:1. The impact of compression ratio on fuel consumption, combustion pressures and exhaust gas emissions has been investigated and presented. Optimum compression ratio which gives best performance has been identified. The results indicate longer ignition delay, maximum rate of pressure rise, lower heat release rate and higher mass fraction burnt at higher compression ratio for pongamia oil methyl ester when compared to that of diesel. The brake thermal efficiency for pongamia oil methyl ester blends and diesel has been calculated and the blend B20 is found to give maximum thermal efficiency. The blends when used as fuel results in reduction of carbon monoxide, hydrocarbon and increase in nitrogen oxides emissions. PME as an oxygenated fuel generated more complete combustion, which means increased torque and power. This is also supported with higher thermal efficiencies of the PME blends. NOx is slightly increased due to the higher combustion temperature and the presence of fuel oxygen with the blend at full load. PME as a new Biodiesel and its blends can be used in diesel engines without any engine modification.

Tribological Investigation and the Effect of Karanja Biodiesel on Engine Wear in Compression Ignition Engine

Year: 2014 Volume: 8 Issue: 2 489 - 494 Pages

Abstract: Various biomass based resources, which can be used as an extender, or a complete substitute of diesel fuel may have very significant role in the development of agriculture, industrial and transport sectors in the energy crisis. Use of Karanja oil methyl ester biodiesel in a CI DI engine was found highly compatible with engine performance along with lower exhaust emission as compared to diesel fuel but with slightly higher NOx emission and low wear characteristics. The combustion related properties of vegetable oils are somewhat similar to diesel oil. Neat vegetable oils or their blends with diesel, however, pose various long-term problems in compression ignition engines. These undesirable features of vegetable oils are because of their inherent properties like high viscosity, low volatility, and polyunsaturated character. Pongamia methyl ester (PME) was prepared by transesterification process using methanol for long term engine operations. The physical and combustion-related properties of the fuels thus developed were found to be closer to that of the diesel. A neat biodiesel (PME) was selected as a fuel for the tribological study of biofuels. Two similar new engines were completely disassembled and subjected to dimensioning of various vital moving parts and then subjected to long-term endurance tests on neat biodiesel and diesel respectively. After completion of the test, both the engines were again disassembled for physical inspection and wear measurement of various vital parts. The lubricating oil samples drawn from both engines were subjected to atomic absorption spectroscopy (AAS) for measurement of various wear metal traces present. The additional lubricating property of biodiesel fuel due to higher viscosity as compared to diesel fuel resulted in lower wear of moving parts and thus improved the engine durability with a bio-diesel fuel. Results reported from AAS tests confirmed substantially lower wear and thus improved life for biodiesel operated engines.

Waste to Biofuel by Torrefaction Technology

Year: 2014 Volume: 8 Issue: 6 549 - 552 Pages

Abstract: Torrefaction is one of waste to energy (WTE) technologies developing in Taiwan recently, which can reduce the moisture and impuritiesand increase the energy density of biowaste effectively.To understand the torrefaction characteristics of different biowaste and the influences of different torrefaction conditions, four typical biowaste were selected to carry out the torrefaction experiments. The physical and chemical properties of different biowaste prior to and after torrefaction were analyzed and compared. Experimental results show that the contents of elemental carbon and caloric value of the four biowaste were significantly increased after torrefaction. The increase of combustible and caloric value in bamboo was the greatest among the four biowaste. The caloric value of bamboo can be increased from 1526 kcal/kg to 6104 kcal/kg after 300oC and 1 hour torrefaction. The caloric valueof torrefied bamboo was almost four times as the original. The increase of elemental carbon content in wood was the greatest (from 41.03% to 75.24%), and the next was bamboo (from 47.07% to 74.63%). The major parameters which affected the caloric value of torrefied biowaste followed the sequence of biowaste kinds, torrefaction time, and torrefaction temperature. The optimal torrefaction conditions of the experiments were bamboo torrefied at 300oC for 3 hours, and the corresponding caloric value of torrefied bamboo was 5953 kcal/kg. This caloric value is similar to that of brown coal or bituminous coal.

A Carbon Footprint Analysis of Rapeseed Oil and Rapeseed Methyl Ester Produced in Romania as Fuels for Diesel Engines

Year: 2013 Volume: 7 Issue: 9 644 - 648 Pages

Abstract: Considering the increasing need of biofuels in Europe and the legislative requirements of the European Union it is needed to quantify the greenhouse gas emissions of biofuels life cycle. In this article a carbon footprint analysis to quantify these gases emitted during production and use of Romanian rapeseed oil (RO) and biodiesel from rapeseed oil (RME) was conducted. The functional unit was considered the LHV of diesel oil of 42.8 MJ·kg-1 corresponding to 1.15kg. of RO and 1.10 kg. of RME. When the 3 fuels were compared, the results show important benefits when using rapeseed oil or biodiesel instead of diesel. The most impacting stage in terms of GHG emissions is the use of the fuels. In this stage, rapeseed oil registers a total quantity of 3,229 kg CO2eq.·FU-1 and biodiesel register a total quantity of 3,088 kg CO2eq.·FU-1 while mineral diesel registers a total quantity of 3,156 kg CO2eq.·FU-1 emitted in the air. Taking into account that rape plant absorbed during growth stage the same quantity of CO2 as emitted into atmosphere during usage stage of the fuel, when compared the three fuels, rapeseed oil and biodiesel obtain obvious benefits against fossil diesel. Results show that by substituting diesel with RO a total quantity of 5,663 kg. CO2eq.·FU-1 would be saved while using biodiesel a total quantity of 5,570 kg. CO2eq.·FU-1 can be saved.

Hydrogen and Biofuel Production from 2-Propanol Over Ru/Al2O3 Catalyst in Supercritical Water

Year: 2013 Volume: 7 Issue: 9 704 - 707 Pages

Abstract: Hydrogen is an important chemical in many industries and it is expected to become one of the major fuels for energy generation in the future. Unfortunately, hydrogen does not exist in its elemental form in nature and therefore has to be produced from hydrocarbons, hydrogen-containing compounds or water. Above its critical point (374.8oC and 22.1MPa), water has lower density and viscosity, and a higher heat capacity than those of ambient water. Mass transfer in supercritical water (SCW) is enhanced due to its increased diffusivity and transport ability. The reduced dielectric constant makes supercritical water a better solvent for organic compounds and gases. Hence, due to the aforementioned desirable properties, there is a growing interest toward studies regarding the gasification of organic matter containing biomass or model biomass solutions in supercritical water. In this study, hydrogen and biofuel production by the catalytic gasification of 2-Propanol in supercritical conditions of water was investigated. Ru/Al2O3 was the catalyst used in the gasification reactions. All of the experiments were performed under a constant pressure of 25 MPa. The effects of five reaction temperatures (400, 450, 500, 550 and 600oC) and five reaction times (10, 15, 20, 25 and 30 s) on the gasification yield and flammable component content were investigated.

Utilization of Laser-Ablation Based Analytical Methods for Obtaining Complete Chemical Information of Algae

Year: 2012 Volume: 6 Issue: 12 1158 - 1162 Pages

Abstract: Themain goal of this article is to find efficient methods for elemental and molecular analysis of living microorganisms (algae) under defined environmental conditions and cultivation processes. The overall knowledge of chemical composition is obtained utilizing laser-based techniques, Laser- Induced Breakdown Spectroscopy (LIBS) for acquiring information about elemental composition and Raman Spectroscopy for gaining molecular information, respectively. Algal cells were suspended in liquid media and characterized using their spectra. Results obtained employing LIBS and Raman Spectroscopy techniques will help to elucidate algae biology (nutrition dynamics depending on cultivation conditions) and to identify algal strains, which have the potential for applications in metal-ion absorption (bioremediation) and biofuel industry. Moreover, bioremediation can be readily combined with production of 3rd generation biofuels. In order to use algae for efficient fuel production, the optimal cultivation parameters have to be determinedleading to high production of oil in selected cellswithout significant inhibition of the photosynthetic activity and the culture growth rate, e.g. it is necessary to distinguish conditions for algal strain containing high amount of higher unsaturated fatty acids. Measurements employing LIBS and Raman Spectroscopy were utilized in order to give information about alga Trachydiscusminutus with emphasis on the amount of the lipid content inside the algal cell and the ability of algae to withdraw nutrients from its environment and bioremediation (elemental composition), respectively. This article can serve as the reference for further efforts in describing complete chemical composition of algal samples employing laserablation techniques.

The Influence of Biofuels on the Permeability of Sand-Bentonite Liners

Year: 2012 Volume: 6 Issue: 12 823 - 826 Pages

Abstract: Liners are made to protect the groundwater table from the infiltration of leachate which normally carries different kinds of toxic materials from landfills. Although these liners are engineered to last for long period of time; unfortunately these liners fail; therefore, toxic materials pass to groundwater. This paper focuses on the changes of the hydraulic conductivity of a sand-bentonite liner due to the infiltration of biofuel and ethanol fuel. Series of laboratory tests were conducted in 20-cm-high PVC columns. Several compositions of sand-bentonite liners were tested: 95% sand: 5% bentonite; 90% sand: 10% bentonite; and 100% sand (passed mesh #40). The columns were subjected to extreme pressures of 40 kPa, and 100 kPa to evaluate the transport of alternative fuels (biofuel and ethanol fuel). For comparative studies, similar tests were carried out using water. Results showed that hydraulic conductivity increased due to the infiltration of alternative fuels through the liners. Accordingly, the increase in the hydraulic conductivity showed significant dependency on the type of liner mixture and the characteristics of the liquid. The hydraulic conductivity of a liner (subjected to biofuel infiltration) consisting of 5% bentonite: 95% sand under pressure of 40 kPa and 100 kPa had increased by one fold. In addition, the hydraulic conductivity of a liner consisting of 10% bentonite: 90% sand under pressure of 40 kPa and 100 kPa and infiltrated by biofuel had increased by three folds. On the other hand, the results obtained by water infiltration under 40 kPa showed lower hydraulic conductivities of 1.50×10-5 and 1.37×10-9 cm/s for 5% bentonite: 95% sand, and 10% bentonite: 90% sand, respectively. Similarly, under 100 kPa, the hydraulic conductivities were 2.30×10-5 and 1.90×10-9 cm/s for 5% bentonite: 95% sand, and 10% bentonite: 90% sand, respectively.

Ethanol Production from Sugarcane Bagasse by Means of Enzymes Produced by Solid State Fermentation Method

Year: 2011 Volume: 5 Issue: 11 1075 - 1078 Pages

Abstract: Nowadays there is a growing interest in biofuel production in most countries because of the increasing concerns about hydrocarbon fuel shortage and global climate changes, also for enhancing agricultural economy and producing local needs for transportation fuel. Ethanol can be produced from biomass by the hydrolysis and sugar fermentation processes. In this study ethanol was produced without using expensive commercial enzymes from sugarcane bagasse. Alkali pretreatment was used to prepare biomass before enzymatic hydrolysis. The comparison between NaOH, KOH and Ca(OH)2 shows NaOH is more effective on bagasse. The required enzymes for biomass hydrolysis were produced from sugarcane solid state fermentation via two fungi: Trichoderma longibrachiatum and Aspergillus niger. The results show that the produced enzyme solution via A. niger has functioned better than T. longibrachiatum. Ethanol was produced by simultaneous saccharification and fermentation (SSF) with crude enzyme solution from T. longibrachiatum and Saccharomyces cerevisiae yeast. To evaluate this procedure, SSF of pretreated bagasse was also done using Celluclast 1.5L by Novozymes. The yield of ethanol production by commercial enzyme and produced enzyme solution via T. longibrachiatum was 81% and 50% respectively.

Classification of Soil Aptness to Establish of Panicum virgatum in Mississippi using Sensitivity Analysis and GIS

Year: 2009 Volume: 3 Issue: 6 191 - 197 Pages

Abstract: During the last decade Panicum virgatum, known as Switchgrass, has been broadly studied because of its remarkable attributes as a substitute pasture and as a functional biofuel source. The objective of this investigation was to establish soil suitability for Switchgrass in the State of Mississippi. A linear weighted additive model was developed to forecast soil suitability. Multicriteria analysis and Sensitivity analysis were utilized to adjust and optimize the model. The model was fit using seven years of field data associated with soils characteristics collected from Natural Resources Conservation System - United States Department of Agriculture (NRCS-USDA). The best model was selected by correlating calculated biomass yield with each model's soils-based output for Switchgrass suitability. Coefficient of determination (r2) was the decisive factor used to establish the 'best' soil suitability model. Coefficients associated with the 'best' model were implemented within a Geographic Information System (GIS) to create a map of relative soil suitability for Switchgrass in Mississippi. A Geodatabase associated with soil parameters was built and is available for future Geographic Information System use.

Comparative Life Cycle Assessment of Rapeseed Oil and Biodiesel from Winter Rape Produced in Romania

Year: 2013 Volume: 7 Issue: 8 555 - 560 Pages

Abstract: The environmental performance of rapeseed oil (RO) and rapeseed methyl ester(RME) from winter rape as fuels produced in Romanian agroclimate is analyzed in this paper. The proposed methodology is life cycle assessment (LCA) and takes into consideration the influence of grain production and agroclimatic conditions. This study shows favorable results first for RO and then for RME. When compared to diesel fuel, both studied biofuels show better results in the following impact categories: Abiotic depletion potential (ADP), Ozone layer depletion (ODP) and Photochemical ozone creation potential (POCP).Furthermore, the environmental performance of the two biofuels studied can be improved by changing the type of fertilizer used and also by using biofuels instead of diesel in the field works.

Fuzzy Multi-Criteria Framework for Supporting Biofuels Policy Making

Year: 2011 Volume: 5 Issue: 11 1731 - 1735 Pages

Authors:
Jadwiga R. Ziolkowska

Abstract: In this paper, a fuzzy algorithm and a fuzzy multicriteria decision framework are developed and used for a practical question of optimizing biofuels policy making. The methodological framework shows how to incorporate fuzzy set theory in a decision process of finding a sustainable biofuels policy among several policy options. Fuzzy set theory is used here as a tool to deal with uncertainties of decision environment, vagueness and ambiguities of policy objectives, subjectivities of human assessments and imprecise and incomplete information about the evaluated policy instruments.

Effect of Temperature and Time on Dilute Acid Pretreatment of Corn Cobs

Year: 2012 Volume: 6 Issue: 4 323 - 327 Pages

Abstract: Lignocellulosic materials are new targeted source to produce second generation biofuels like biobutanol. However, this process is significantly resisted by the native structure of biomass. Therefore, pretreatment process is always essential to remove hemicelluloses and lignin prior to the enzymatic hydrolysis. The goals of pretreatment are removing hemicelluloses and lignin, increasing biomass porosity, and increasing the enzyme accessibility. The main goal of this research is to study the important variables such as pretreatment temperature and time, which can give the highest total sugar yield in pretreatment step by using dilute phosphoric acid. After pretreatment, the highest total sugar yield of 13.61 g/L was obtained under an optimal condition at 140°C for 10 min of pretreatment time by using 1.75% (w/w) H3PO4 and at 15:1 liquid to solid ratio. The total sugar yield of two-stage process (pretreatment+enzymatic hydrolysis) of 27.38 g/L was obtained.

Trans-Esterification for Production of Biodiesel from Waste Frying Oil (WFO)

Year: 2010 Volume: 4 Issue: 11 706 - 710 Pages

Abstract: Biodisel is a type of biofuel having similar properties of diesel fuel but lacks substances (undesirable emissions) such as sulfur, nitrogen and aromatic polycyclic. Upon filtration of waste oil, the biodiesel fuel was produced via carrying out transestrification reaction of triglycerides followed by conducting viscosity, density, flash point, cloud point, pour point and copper strip corrosion tests on the samples and comparing with EN14214 and ASTM 6751 standards and all results were found in the permitted limit. The highest yield of biodiesel production reaction was found 46.6435 g when Sodium Hydroxide catalyst in amount of 0.375g was employed, 44.2347 g when Sodium methoxide catalyst in amount of 0.5g was employed and 56.5124 g when acid sulfuric catalyst in amount of 1g was employed and 47.3290 g when two stage reaction was done.

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