Ammonia fiber expansion pretreatment and enzymatic hydrolysis on two different growth stages of reed canarygrass

Plant materials from the vegetative growth stage of reed canarygrass and the seed stage of reed canarygrass are pretreated by ammonia fiber expansion (AFEX) and enzymatically hydrolyzed using 15 filter paper units (FPU) cellulase/g glucan to evaluate glucose and xylose yields. Percent conversions of glucose and xylose, effects of temperature and ammonia loading, and hydrolysis profiles are analyzed to determine the most effective AFEX treatment condition for each of the selected materials. The controls used in this study were untreated samples of each biomass material. All pretreatment conditions tested enhanced enzyme digestibility and improved sugar conversions for reed canarygrass compared with their untreated counterparts. Based on 168 h hydrolysis results using 15 FPU Spezyme CP cellulase/g glucan the most effective AFEX treatment conditions were determined as: vegetative growth stage of reed canarygrass--100 degrees C, 60% moisture content, 1.2:1 kg ammonia/kg of dry matter (86% glucose and 78% xylose) and seed stage of reed canarygrass--100 degrees C, 60% moisture content, 0.8:1 kg ammonia/kg of dry matter (89% glucose and 81% xylose). Supplementation by commercial Multifect 720 xylanase along with cellulase further increased both glucose and xylose yields by 10-12% at the most effective AFEX conditions.     

Pretreatment of switchgrass by ammonia fiber explosion (AFEX)

The effects of ammonia fiber explosion (AFEX) pretreatment of switch grass using its major process variables are reported. The optimal pretreatment conditions for switchgrass were found to be near 100°C reactor temperature, and ammonia loading of 1:1 kg of ammonia: kg of dry matter with 80% moisture content (dry weight basis [dwb]) at 5 min residence time. Hydrolysis results of AFEX-treated and untreated samples showed 93% vs 16% glucan conversion, respectively. The ethanol yield of optimized AFEX-treated switchgrass was measured to be about 0.2 g ethanol/g dry biomass, which is 2.5 times more than that of the untreated sample.     

Enzymatic hydrolysis of high-moisture corn fiber pretreated by afex and recovery and recycling of the enzyme complex

Corn fiber is a grain-processing residue containing significant amounts of cellulose, hemicellulose, and starch, which is collected in facilities where fuel ethanol is currently manufactured. Preliminary research has shown that corn fiber (30% moisture dry weight basis [dwb]) responds well to ammonia-fiber explosion (AFEX) pretreatment. However, an important AFEX pretreatment variable that has not been adequately explored for corn fiber is sample moisture. In the present investigation, we determined the best AFEX operating conditions for pretreatment of corn fiber at high moisture content (150% moisture dwb). The optimized AFEX treatment conditions are defined in terms of the moisture content, particle size, ammonia to biomass ratio, temperature, and residence time using the response of the pretreated biomass to enzymatic hydrolysis as an indicator. Approximate optimal-pretreatment conditions for unground corn fiber containing 150% (dwb) moisture were found to be: temperature, 90?C; ammonia: dry corn fiber mass ratio, 1:1; and residence time 30 min (average reactor pressure under these conditions was 200 pounds per square inch [psig]). Enzymatic hydrolysis of the treated corn fiber was performed with three different enzyme combinations. More than 80% of the theoretical sugar yield was obtained during enzymatic hydrolysis using the best enzyme combination after pretreatment of corn fiber under the optimized conditions previously described. A simple process for enzyme recovery and reuse to hydrolyze multiple portions of AFEX-treated corn fiber by one portion of enzyme preparation is demonstrated. Using this process, five batches of fresh substrate (at a concentration of 5% w/v) were successfully hydrolyzed by repeated recovery and reuse of one portion of enzyme preparation, with the addition of a small portion of fresh enzyme in each subsequent recycling step.     

Hydrolysis of Ammonia-pretreated Sugar Cane Bagasse with Cellulase, β-Glucosidase, and Hemicellulase Preparations

Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH₃: 1 biomass, 100 °C, 30 min) or with NH₄OH (0.5 g NH₄OH of a 28% [v/v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.     

Extraction of Proteins from Switchgrass Using Aqueous Ammonia within an Integrated Biorefinery

Switchgrass (Panicum vergatum) is a potential feedstock for future cellulosic biorefineries. Such a feedstock may also provide protein, most likely for use as an animal feed. In this paper, we present a potential scheme for integrating fiber processing with extractions to obtain both sugar and protein products from switchgrass pretreated using Ammonia Fiber Expansion (AFEX). Solutions of 3% aqueous ammonia at pH 10.5 provided optimal extraction of proteins. Addition of the nonionic surfactant Tween-80 improved protein recovery for AFEX-treated materials. It was determined that an extraction following AFEX solubilized approximately 40% of the protein, while a subsequent hydrolysis solubilized much of the remaining protein while producing 325 g sugar per kg biomass. The remaining insoluble residue contained very little protein or ash, making it ideal for heat and power production. In contrast, an extraction following hydrolysis solubilized only 68% of the original protein in the biomass, while obtaining slightly higher sugar yields.     

Ammonia fiber explosion treatment of corn stover

Optimizing process conditions and parameters such as ammonia loading, moisture content of biomass, temperature, and residence time is necessary for maximum effectiveness of the ammonia fiber explosion process. Approximate optimal pretreatment conditions for corn stover were found to be temperature of 90°C, ammonia: dry corn stover mass ratio of 1∶1, moisture content of corn stover of 60% (dry weight basis), and residence time (holding at target temperature), of 5 min. Approximately 98% of the theoretical glucose yield was obtained during enzymatic hydrolysis of the optimal treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). The ethanol yield from this sample was increased up to 2.2 times over that of untreated sample. Lowering enzyme loading to 15 and 7.5 FPU/g of glucan did not significantly affect the glucose yield compared with 60 FPU, and any differences between effects at different enzyme levels decreased as the treatment temperature increased.     

Pilot-Scale Fermentation of Aqueous-Ammonia-Soaked Switchgrass

Aqueous-ammonia-steeped switchgrass was subject to simultaneous saccharification and fermentation (SSF) in two pilot-scale bioreactors (50- and 350-L working volume). Switchgrass was pretreated by soaking in ammonium hydroxide (30%) with solid to liquid ratio of 5 L ammonium hydroxide per kilogram dry switchgrass for 5 days in 75-L steeping vessels without agitation at ambient temperatures (15 to 33 °C). SSF of the pretreated biomass was carried out using Saccharomyces cerevisiae (D₅A) at approximately 2% glucan and 77 filter paper units per gram cellulose enzyme loading (Spezyme CP). The 50-L fermentation was carried out aseptically, whereas the 350-L fermentation was semiaseptic. The percentage of maximum theoretical ethanol yields achieved was 73% in the 50-L reactor and 52–74% in the 350-L reactor due to the difference in asepsis. The 350-L fermentation was contaminated by acid-producing bacteria (lactic and acetic acid concentrations approaching 10 g/L), and this resulted in lower ethanol production. Despite this problem, the pilot-scale SSF of aqueous-ammonia-pretreated switchgrass has shown promising results similar to laboratory-scale experiments. This work demonstrates challenges in pilot-scale fermentations with material handling, aseptic conditions, and bacterial contamination for cellulosic fermentations to biofuels.     

Extrusion processing for ammonia fiber explosion (AFEX)

The ammonia fiber explosion (AFEX) process, previously run only in a batch reactor, has been adapted to run on a twin-screw extruder. The sugar yield of AFEX material after enzymatic hydrolysis has been increased up to 3.5 times over that of completely untreated material. The ruminant digestibility of corn fodder has been increased up to 32% (from 54–71%) over completely untreated material, and 23% (from 63–77%) over material extruded with no ammonia. Extrusion-treated material proved more digestible by the ruminant at 48 h than material treated in the batch reactor.     

Ethanol production from enzymatic hydrolysates of AFEX-treated coastal bermudagrass and switchgrass

Applied Biochemistry and Biotechnology - Switchgrass and coastal bermudagrass were pretreated by ammonia fiber explosion (AFEX), and the treated materials hydrolyzed using 5 IU cellulase/g...     

Kinetics of Lime Pretreatment of Sugarcane Bagasse to Enhance Enzymatic Hydrolysis

The objective of this work was to determine the optimum conditions of sugarcane bagasse pretreatment with lime to increase the enzymatic hydrolysis of the polysaccharide component and to study the delignification kinetics. The first stage was an evaluation of the influence of temperature, reaction time, and lime concentration in the pretreatment performance measured as glucose release after hydrolysis using a 2³ central composite design and response surface methodology. The maximum glucose yield was 228.45 mg/g raw biomass, corresponding to 409.9 mg/g raw biomass of total reducing sugars, with the pretreatment performed at 90°C, for 90 h, and with a lime loading of 0.4 g/g dry biomass. The enzymes loading was 5.0 FPU/dry pretreated biomass of cellulase and 1.0 CBU/dry pretreated biomass of β-glucosidase. Kinetic data of the pretreatment were evaluated at different temperatures (60°C, 70°C, 80°C, and 90°C), and a kinetic model for bagasse delignification with lime as a function of temperature was determined. Bagasse composition (cellulose, hemicellulose, and lignin) was measured, and the study has shown that 50% of the original material was solubilized, lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (60–90°C). The delignification was highly dependent on temperature and duration of pretreatment.     

Lime pretreatment of crop residues bagasse and wheat straw

Lime (calcium hydroxide) was used as a pretreatment agent to enhance the enzymatic digestibility of two common crop residues: bagasse and wheat straw. A systematic study of pretreatment conditions suggested that for short pretreatment times (1–3 h), high temperatures (85-135°C) were required to achieve high sugar yields, whereas for long pretreatment times (e.g., 24 h), low temperatures (50–65°C) were effective. The recommended lime loading is 0.1 g Ca(OH)₂/g dry biomass. Water loading had little effect on the digestibility. Under the recommended conditions, the 3-d reducing sugar yield of the pretreated bagasse increased from 153 to 659 mg Eq glucose/g dry biomass, and that of the pretreated wheat straw increased from 65 to 650 mg Eq glucose/g dry biomass. A material balance study on bagasse showed that the biomass yield after lime pretreatment is 93.6%. No glucan or xylan was removed from bagasse by the pretreatment, whereas 14% of lignin became solubilized. A lime recovery study showed that 86% of added calcium was removed from the pretreated bagasse by ten washings and could be recovered by carbonating the wash water with CO₂ at pH 9.5.     

Cost estimates and sensitivity analyses for the ammonia fiber explosion process

Process designs were conducted for each unit of the conceptual ammonia fiber explosion (AFEX) process, and fixed capital investment and operating costs were estimated. AFEX costs about $20-40/t of dry bio-mass treated. Several promising areas for reducing process costs exist. Return on investment (ROI) calculations were also done for AFEX-treated materials (as digestibility-enhanced animal feeds), in conjunction with sensitivity analyses on the overall processing costs. Estimated ROIs range from over 100%/y to negative, depending on the system variables. The most important variables are the cost of corn and corn fiber, ammonia loading, and whether or not drying is required.     

Integrated production of ethanol fuel and protein from Coastal Bermudagrass

The herbaceous crops that may provide fermentable carbohydrates for production of fuels and chemicals also contain 10–20% protein. Protein coproduction with biomass-derived fuels and chemicals has important advantages: (1) food and fuel production can be integrated, and (2) protein is a high-value product that may significantly improve overall process economics. We report the results of an integrated approach to producing protein and fermentable sugars from one herbaceous species, Coastal Bermudagrass (CBG). The ammonia fiber explosion (AFEX) process makes possible over 90% conversion of cellulose and hemicellulose to simple sugars (about 650 mg reducing sugars/g dry CBG) at 5 IU cellulase/g vs < 20% conversion for untreated CBG. The AFEX treatment also improves protein extraction from CBG; over 80% protein recovery is possible from AFEX-treated CBG vs about 30% recovery from untreated CBG.     

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R ² > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.     

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatmett or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60°C, 0.5∶1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.     

Fermentation of Sugarcane Bagasse and Chicken Manure to Calcium Carboxylates under Thermophilic Conditions

Sugarcane bagasse and chicken manure were anaerobically fermented to carboxylic acids using a mixed culture of marine microorganisms at 55 °C. Using the MixAlco process— an example of consolidated bioprocessing— the resulting carboxylate salts can be converted to mixed alcohol fuels or gasoline. To enhance digestibility, sugarcane bagasse was lime pretreated with 0.1 g Ca(OH)₂/g dry biomass at 100 °C for 2 h. Four-stage countercurrent fermentation of 80% sugarcane bagasse/20% chicken manure was performed at various volatile solids (VS) loading rates and liquid residence times. Calcium carbonate was used as a buffer during fermentation. The highest acid productivity of 0.79 g/(L day) occurred at a total acid concentration of 21.5 g/L. The highest conversion (0.59 g VS digested/g VS fed) and yield (0.18 g total acids/g VS fed) occurred at a total acid concentration of 15.5 g/L. The continuum particle distribution model (CPDM) predicted the experimental total acid concentrations and conversions at an average error of 10.14% and 12.68%, respectively. CPDM optimizations show that high conversion (>80%) and total acid concentration of 21.3 g/L are possible with 300 g substrate/(L liquid), 30 days liquid residence time, and 3 g/(L day) solid loading rate. Thermophilic fermentation has a higher acetate content (∼63 wt%) than mesophilic fermentation (∼39 wt%).     

Enhanced Enzymatic Hydrolysis of Lignocellulose by Optimizing Enzyme Complexes

To enhance the conversion of the cellulose and hemicellulose, the corncob pretreated by aqueous ammonia soaking was hydrolyzed by enzyme complexes. The saturation limit for cellulase (Spezyme CP) was determined as 15 mg protein/g glucan (50 filter paper unit (FPU)/g glucan). The accessory enzymes (β-glucosidase, xylanase, and pectinase) were supplemented to hydrolyze cellobiose (cellulase-inhibiting product), hemicellulose, and pectin (the component covering the fiber surfaces), respectively. It was found that β-glucosidase (Novozyme 188) loading of 1.45 mg protein/g glucan [30 cellobiase units (CBU)/g glucan] was enough to eliminate the cellobiose inhibitor, and 2.9 mg protein/g glucan (60 CBU/g glucan) was the saturation limit. The supplementation of xylanase and pectinase can increase the conversion of cellulose and hemicellulose significantly. The yields of glucose and xylose enhanced with the increasing enzyme loading, but the increasing trend became low at high loading. Compared with xylanase, pectinase was more effective to promote the hydrolysis of cellulose and hemicellulose. The supplementation of pectinase with 0.12 mg protein/g glucan could increase the yields of glucose and xylose by 7.5% and 29.3%, respectively.     

Bioethanol Production Involving Recombinant C. thermocellum Hydrolytic Hemicellulase and Fermentative Microbes

The enhancement of the biomass productivity of Escherichia coli cells harbouring the truncated 903?bp gene designated as glycoside hydrolase family 43 (GH43) from Clostridium thermocellum showing hemicellulase activity along with its further use in simultaneous saccharification and fermentation (SSF) process is described. (Phosphoric acid) H₃PO₄?Cacetone treatment and ammonia fibre expansion (AFEX) were the pretreatment strategies employed on the leafy biomass of mango, poplar, neem and asoka among various substrates owing to their high hemicellulose content. GH43 showed optimal activity at a temperature of 50?°C, pH?5.4 with stability over a pH range of 5.0?C6.2. A 4-fold escalation in growth of the recombinant E. coli cells was observed when grown using repeated batch strategy in LB medium supplemented with glucose as co-substrate. Candida shehatae utilizing pentose sugars was employed for bioethanol production. AFEX pretreatment proved to be better over acid?Cacetone technique. The maximum ethanol concentration (1.44?g/L) was achieved for AFEX pretreated mango (1%, w/v) followed by poplar with an ethanol titre (1.32?g/L) in shake flask experiments. A 1.5-fold increase in ethanol titre (2.11?g/L) was achieved with mango (1%, w/v) in a SSF process using a table top 2-L bioreactor with 1?L working volume.     

Ion-pair hollow-fiber liquid-phase microextraction of the quaternary ammonium surfactant dicocodimethylammonium chloride

A two-phase hollow-fiber (HF) liquid-phase microextraction (LPME) method was developed for determination of a quaternary ammonium compound surfactant, dicocodimethylammonium chloride, in aqueous samples. The porous HF was fixed on a metal rod support and was impregnated with approximately 6.6 μL of organic extractant, which was immobilized in the HF pores. Surfactant extraction was facilitated by addition of carboxylic acid to the sample forming neutral ion pairs with the quaternary ammonium compound. After extraction, the analyte was transferred from the organic extractant in the fiber pores by dissolving the 1-octanol into 100 μL methanol. The methanol extract was analyzed by liquid chromatography–mass spectrometry. The method was optimized (with optimized parameters in brackets) with regard to type of organic extractant (1-octanol), fiber length (2 cm), choice and concentration of anionic carrier (600 μg L⁻¹ octanoate), procedure of transfer to methanol (15-min sonication), sample volume (250 mL), extraction time (17 h), pH (10), and ionic strength (50 mM carbonate). Aspects influencing repeatability in LPME of (quaternary ammonium) surfactants are discussed. The enrichment factor achieved in 250-mL carbonate buffer was around 400. Due to matrix effects, the enrichment factors achieved when industrial process water was analyzed were 120 or about 30% of that in carbonate buffer. Detection limits of 0.3 μg L⁻¹ in carbonate buffer and 0.9 μg L⁻¹ in industrial process water were obtained. If the studied compound is seen as a model substance representing quaternary dialkylated dimethylated ammonium surfactants in general, the developed method may be applied to other quaternary ammonium surfactants.     

Heterotrophic Culture of <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> in Various Nitrogen Sources for Lipid Production

The influences of urea, yeast extract, and nitrate as the nitrogen source on heterotrophic growth of four strains of Chlorella protothecoides were investigated in 9-day feed-batch cultures. Biomass dry weight concentration (DWC) and lipid yield (LY) of the four strains in all media were compared. The highest LY in 9 days was 654 mg/L/day by UTEX 255 in 2.4 g/L KNO₃ medium with a biomass DWC of 11.7 g/L and lipid content of 50.5%. Using green autotrophic seeds instead of yellow heterotrophic seeds improved the biomass DWC (13.1 vs. 11.7 g/L), LY (850 vs. 654 mg/L/day), and lipid to glucose consumption ratio (0.607 vs. 0.162). Moreover, 17.0 g/L DWC and 489 mg/L/day LY were obtained from the sequentially mixed-nitrogen medium, and the lipid to glucose consumption ratio was improved to 0.197 from 0.162 in 2.4 g/L nitrate medium and from 0.108 in 4.2 g/L yeast extract medium in the first batch.