Scale-up study of oxalic acid pretreatment of agricultural lignocellulosic biomass for the production of bioethanol

Building on our laboratory-scale optimization, oxalic acid was used to pretreat corncobs on the pilot-scale. The hydrolysate obtained after washing the pretreated biomass contained 32.55 g/l of xylose, 2.74 g/l of glucose and low concentrations of inhibitors. Ethanol production, using Scheffersomyces stipitis, from this hydrolysate was 10.3 g/l, which approached the predicted value of 11.9 g/l. Diafiltration using a membrane system effectively reduced acetic acid in the hydrolysate, which increased the fermentation rate. The hemicellulose content of the recovered solids decreased from 27.86% before pretreatment to only 6.76% after pretreatment. Most of the cellulose remained in the pretreated biomass. The highest ethanol production after simultaneous saccharification and fermentation (SSF) of washed biomass with S. stipitis was 21.1 g/l.     

Preparation of carbon black from rice husk by hydrolysis, carbonization and pyrolysis

Carbon black is a form of amorphous carbon that is produced by incomplete combustion of petroleum- or some plant-derived materials and has a number of industrial uses. A process consisting of hydrolysis, carbonization and pyrolysis of rice husk was developed. Under optimal hydrolysis conditions (72 wt.% sulfuric acid, 50 °C, 10 min), a hydrolysis ratio of 52.72% was achieved. After carbonization of the hydrolysis solution by water bath, the solid carbon was further pyrolyzed. As the pyrolysis temperature was increased from 400 to 800 °C, the carbon content increased from 83.41% to 94.66%, the number of O-H, C-H, CO, and CC surface functional groups decreased, and based on Brunauer-Emmett-Teller (BET) results, the specific surface area and pore volume of carbon black increased from 389 to 1034 m²/g and from 0.258 to 0.487 cm³/g, respectively. X-ray diffraction pattern (XRD) and Raman spectroscopy analyses of samples pyrolyzed at 400-800 °C showed a localized graphitic structure. It is possible that the hydrolysis/carbonization/pyrolysis process developed in this study could also be applicable to the preparation of carbon black from other types of biomass.     

Effect of pretreatment severity on the conversion of barley straw to fermentable substrates and the release of inhibitory compounds

The production of fermentable substrates from barley straw under various process conditions was studied. Pretreatment included chemical pretreatment with dilute-acid followed by enzymatic hydrolysis; the pretreatment conditions were expressed in a combined severity factor, CS, which ranged in the present study from −1.6 to 1.1. Considering the production of fermentable sugars and the release of inhibitory compounds, the optimal pretreatment conditions were 170 °C, 0% sulfuric acid and 60 min, corresponding to CS −0.4. Under these conditions, 21.4 g glucose/L, 8.5 g xylose/L, and 0.5 g arabinose/L were produced, while 0.1 g HMF/L, 0.4 g furfural/L, 0.0 g levulinic acid/L, 0.0 g formic acid/L, and 2.1 g acetic acid/L were released. The ratio of Σsugars/Σinhibitors proved to be a good tool for evaluating the suitability of a hydrolysate for fermentation purposes.     

Bio-oil from hydro-liquefaction of Dunaliella salina over Ni/REHY catalyst

The hydro-liquefaction of Dunaliella salina over solid acid catalyst was examined under moderate conditions (200 °C, 2.0 MPa, 60 min). The significant increment of bio-oil yield was obtained over Ni/REHY catalyst, increasing about 20% compared without modified REHY. H₂-temperature-programmed desorption (H₂-TPD) and X-ray powder diffraction (XRD) demonstrated that Ni/REHY as bifunctional catalyst played roles in hydrogenation and cracking, and further achieved the deoxygenation and desulfurization of D. salina under hydrogen gas. The oxygen and sulfur contents of bio-oils decreased relative to D. salina, and the higher heating value (HHV) significantly increased to 30.11 MJ/kg. From gas chromatography/mass spectrometry (GC/MS), the dominant compounds of the bio-oils were found to be esters, glycerins. The qualities of bio-oils were greatly improved. Hydro-liquefaction was beneficial to the exploration of bio-oils from microalgae.     

Influences of polyunsaturated fatty acids (PUFAs) on growth and secondary metabolite accumulation in Panax ginseng C.A. Meyer adventitious roots cultured in air-lift bioreactors

The present study relates to different polyunsaturated fatty acids (PUFAs) which were used as elicitors to enhance biomass accumulation and ginsenoside production in Panax ginseng. Adventitious root cultures of ginseng were elicited with oleic and linolenic acid at 0, 1, 5, 10 or 50 µmol/l concentrations respectively. Elicitors were added to the medium of adventitious roots on the 40th day of culture and roots were harvested on day 47. Cultures supplemented with oleic acid decreased root biomass and ginsenoside accumulation. Cultures supplemented with 1 µmol/l linolenic acid enhanced ginsenoside accumulation, without the decrease of adventitious root biomass. Linolenic acid enhanced the biosynthesis of both protopanxatriols (2.95 ± 0.048 mg/g DW) and protopanxadiols (5.66 ± 0.043 mg/g DW) compared to that of control at (1.41 ± 0.002 mg/g DW) and (1.58 ± 0.006 mg/g DW) respectively. No changes in polysaccharides and phenolics content have been noticed upon elicitation with PUFAs. This is the first report on linolenic acid as an elicitor for ginsenoside accumulation in ginseng adventitious root cultures.     

Laboratory scale examination of the effects of overloading on the anaerobic digestion by glycerol

The anaerobic digestion of pure glycerol, which produces a baseline acetic acid to propionic acid ratio of 0.2, was studied in laboratory scale reactors (3 l working volume) at mesophilic temperature (37 °C) with 3000 mg chemical oxygen demand (COD) l⁻¹d⁻¹. During the experiment tVFA and C2-C6 VFA analysis and daily biogas yield measurement were carried out. Following 10 days of a 15% d⁻¹ increase in the organic loading rate (OLR) of 3.0-10.5 g COD l⁻¹d⁻¹, the concentration of propionic acid increased to 6200-8000 mg l⁻¹. Then the inoculum was divided into three parts feeding with 100% glycerol, 50% glycerol + 50% acetic acid, and 50% glycerol + 50% thick stillage, (presented in % of 2.60 g COD l⁻¹d⁻¹ OLR), respectively. The application of co-substrates reduced the recovery period by 5 days compared to feeding with pure glycerol. When the reactors were loaded with glycerol again (10% OLR raise per day) the previously applied co-substrates had a positive effect on the VFA composition and the biogas yield as well.     

Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol production by altering lignin composition

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cultivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-type control. Both were harvested at two maturities. All the samples had similar carbohydrate contents including a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield of 382 l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pretreated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232-278 l/ton and were significantly enhanced for the reduced S lignin cultivars.     

Somatic embryo mediated mass production of Catharanthus roseus in culture vessel (bioreactor) – A comparative study

The purpose of this study was to evaluate and compare the use of liquid and solid Murashige and Skoog (MS) medium in different culture vessels for mass production of Catharanthus roseus, an important source of anticancerous compounds, vincristine and vinblastine. Three media conditions i.e. agar-solidified medium (S), liquid medium in agitated conical flask (L) and growtek bioreactor (B) were used. Rapid propagation was achieved through in vitro somatic embryogenesis pathway. The process of embryogenesis has been categorized into induction, proliferation, maturation and germination stages. All in vitro embryogenesis stages were conducted by withdrawing spent liquid medium and by adding fresh MS medium. In optimized 4.52 μM 2,4-D added MS, the callus biomass growth was low in solid (1.65 g) compared to liquid medium in agitated conical flask (1.95 g) and in bioreactor (2.11 g). The number of normal somatic embryos was more in solid medium (99.75/50 mg of callus mass) compared to liquid medium used in conical flask (83.25/callus mass) and growtek bioreactor (84.88/callus mass). The in vitro raised embryos maturated in GA₃ (2.60 μM) added medium; and in bioreactor the embryo growth was high, a maximum length of 9.82 mm was observed at the end of four weeks. These embryos germinated into seedlings in BAP (2.22 μM) added medium and the embryo germination ability was more (59.41%) in bioreactor compared to liquid medium in conical flask (55.5%). Shoot length (11.25 mm) was also high in bioreactor compared to agitated conical flask. The liquid medium used in agitated conical flask and bioreactor increased seedling production efficiency, at the same time it also reduced plant recovery time. The embryo generated plants grew normally in outdoor conditions. The exploitation of medium to large culture vessel or bioreactor may make the process more efficient in getting large number of Catharanthus plant as it is the only source of anti-cancerous alkaloids, vincristine and vinblastine.Abbreviations: BA, N6-benzyladenine; 2,4-D, 2,4-Dichlorophenoxyacetic acid; GA₃, gibberellic acid; NAA, naphthalene acetic acid; MS, Murashige and Skoog (1962) medium; S, agar-solidified medium; L, liquid medium in agitated conical flask; B, growtek bioreactor     

From residual to useful oil: Revalorization of glycerine from the biodiesel synthesis

The reaction kinetics for the synthesis of glycerol triacetate (triacetin) from glycerol and acetic acid has been studied in the frame of revalorization of residual glycerol in biodiesel production. The reaction has taken place in a stirred reactor at a pressure of 1070 kPa. No external catalyst has been added because the reaction performs better by using as catalyst an excess of acetic acid. Kinetic parameters were obtained at 120 and 160 °C and a model of three reactions with monoacetin, diacetin and triacetin formation was proposed with a good agreement with the experimental results. Arrhenius constants were determined for the involved reactions.     

Catalytic hydrolysis of lignocellulosic biomass into 5-hydroxymethylfurfural in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) from cellulose catalyzed by solid acids and metal chlorides was studied in the 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) under microwave irradiation. Among the applied catalysts, the use of CrCl₃/LiCl resulted in the highest yield of HMF. The effects of catalyst dosage (mole ratio of catalyst to glucose units in the feedstock) and reaction temperature on HMF yields were investigated to obtain optimal process conditions. With the 1:1 mol ratio of catalyst to glucose unit, the HMF yield reached 62.3% at 160 °C for 10 min. Untreated wheat straw was also investigated as feedstock to produce HMF for the practical use of raw biomass, in which the HMF yield was comparable to that from pure cellulose. After the extraction of HMF, [BMIM]Cl and CrCl₃/LiCl could be reused and exhibited no activity loss after three successive runs.     

Effect of reduced sulfur compounds on the fermentation of phosphoric acid pretreated sugarcane bagasse by ethanologenic Escherichia coli

The addition of reduced sulfur compounds (thiosulfate, cysteine, sodium hydrosulfite, and sodium metabisulfite) increased growth and fermentation of dilute acid hydrolysate of sugarcane bagasse by ethanologenic Escherichia coli (strains LY180, EMFR9, and MM160). With sodium metabisulfite (0.5 mM), toxicity was sufficiently reduced that slurries of pretreated biomass (10% dry weight including fiber and solubles) could be fermented by E. coli strain MM160 without solid-liquid separation or cleanup of sugars. A 6-h liquefaction step was added to improve mixing. Sodium metabisulfite also caused spectral changes at wavelengths corresponding to furfural and soluble products from lignin. Glucose and cellobiose were rapidly metabolized. Xylose utilization was improved by sodium metabisulfite but remained incomplete after 144 h. The overall ethanol yield for this liquefaction plus simultaneous saccharification and co-fermentation process was 0.20 g ethanol/g bagasse dry weight, 250 L/tonne (61 gal/US ton).     

Effects of Agave salmiana Otto Ex Salm-Dyck silage as forage on ruminal fermentation and growth in goats

Ensiling of Agave salmiana Otto Ex Salm-Dyck, a widespread plant in Mexico, as a viable preservation method to create a potential animal feed resource for ruminants was investigated. Fresh A. salmiana with 205 g dry matter (DM)/kg and wilted alfalfa with 602 g DM/kg were ensiled in combinations (DM:DM) of 1000:0, 500:500 and 350:650, to evaluate feeding value of agave:alfalfa silages on ruminal fermentation and growth of goats. Chemical composition and in situ ruminal disappearance of three total mixed rations (TMRs), which included 240 g/kg DM of each silage (1000:0, 500:500 and 350:650) were determined. The TMR were used to assess ruminal fermentation and growth of 15 goats (20 ± 2.2 kg body weight (BW)). Silage pH (≤4), lactate (>25 g/kg DM) and ammonia (<50 g/kg total N) concentrations indicate that silage quality was good. Lactic acid was the main acid in all silages, acetic acid concentrations were relatively low, and butyrate was only detected in only the 1000:0 agave:alfalfa silage. Potential DM disappearance of the TMR increased quadratically as the amount of alfalfa included in the silage mixture increased. The BW gain and feed efficiency were not changed by treatment, even though DM intake decreased and aNDF intake increased linearly as the amount of alfalfa included in the silage mixture increased. Ruminal pH and butyrate increased, and ammonia N, lactate and propionate decreased linearly as alfalfa proportion of alfalfa in the silage mixture was increased. The TMR ingredient selectivity by the goats may have limited goat performance when alfalfa was included in agave silage mixtures. Because the agave:alfalfa blend improved nutritional quality, ruminal digestibility and intake of agave silage, alfalfa inclusion may improve nutritional characteristics of agave plants silages for ruminants.     

Effects of different pretreatment strategies on corn stalk acidogenic fermentation using a microbial consortium

The effects of sulfuric acid, acetic acid, aqueous ammonia, sodium hydroxide, and steam explosion pretreatments of corn stalk on organic acid production by a microbial consortium, MC1, were determined. Steam explosion resulted in a substrate that was most favorable for microbial growth and organic acid productions. The total amounts of organic acids produced by MC1 on steam exploded, sodium hydroxide, sulfuric acid, acetic acid, and aqueous ammonia pretreated corn stalk were 2.99, 2.74, 1.96, 1.45, and 2.21 g/l, respectively after 3 days of fermentation at 50 °C. The most prominent organic products during fermentation of steam-exploded corn stalks were formic (0.86 g/l), acetic (0.59 g/l), propanoic (0.27 g/l), butanoic (0.62 g/l), and lactic acid (0.64 g/l) after 3 days of fermentation; ethanol (0.18 g/l), ethanediol (0.68 g/l), and glycerin (3.06 g/l) were also produced. These compounds would be suitable substrates for conversion to methane by anaerobic digestion.     

Hydrothermal liquefaction of the brown macro-alga Laminaria saccharina: effect of reaction conditions on product distribution and composition

The brown macro-alga Laminaria saccharina was converted into bio-crude by hydrothermal liquefaction in a batch reactor. The influence of reactor loading, residence time, temperature and catalyst (KOH) loading was assessed. A maximum bio-crude yield of 19.3 wt% was obtained with a 1:10 biomass:water ratio at 350 °C and a residence time of 15 min without the presence of the catalyst. The bio-crude had an HHV of 36.5 MJ/kg and is similar in nature to a heavy crude oil or bitumen. The solid residue has high ash content and contains a large proportion of calcium and magnesium. The aqueous phase is rich in sugars and ammonium and contains a large proportion of potassium and sodium.     

Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment

Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3⁴) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120 °C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20 PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40 °C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9 × 10¹¹ CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS.     

Pretreatment of switchgrass for sugar production with the combination of sodium hydroxide and lime

Sodium hydroxide (NaOH) and lime (Ca(OH)₂) were innovatively used together in this study to improve the cost-effectiveness of alkaline pretreatment of switchgrass at ambient temperature. Based on the sugar production in enzymatic hydrolysis, the best pretreatment conditions were determined as: residence time of 6 h, NaOH loading of 0.10 g/g raw biomass, NaOH addition at the beginning, Ca(OH)₂ loading of 0.02 g/g raw biomass, and biomass wash intensity of 100 ml water/g raw biomass, at which the glucose and xylose yields were respectively 59.4% and 57.3% of the theoretical yields. The sugar yield of the biomass pretreated using the combination of 0.10 g NaOH/g raw biomass and 0.02 g Ca(OH)₂/g raw biomass was found comparable with that of the biomass pretreated using 0.20 g NaOH/g raw biomass at the same conditions, while the chemical expense was remarkably reduced due to the low cost of lime and the reduced loading of NaOH.     

Assessment of two biomass estimation methods for aquatic vegetation growing on the Amazon Floodplain

Studies of macrophyte productivity in the Amazon region are limited by accessibility and costs; hence, they may suffer from reduced sample size and representation. The present study compares a phenometric (indirect) method and a subsampling (direct) method in terms of accuracy and applicability to estimation of aquatic macrophyte biomass in the Amazon. The results show that phenometric models were not as effective as selective subsampling for the estimation of macrophyte biomass under the studied conditions. Phenometric models performed more acceptably for predicting emergent biomass, and less for submerged and total biomass (r² = 0.77, p < 0.05, RMSE = 200-600 g/m² dry mass). Improvements in r² by using species-specific phenometric models were mostly not significant. Phenotypic variation across the studied region was large enough to preclude the generalization of phenometric relationships into accurate numeric models, while the direct subsampling method was able to account for this variation (RMSE < 500 g/m² dry mass). Subsampling also allowed a significant reduction on the physical effort of biomass sampling, which directly translated into wider and more complete sampling. We suggest that direct subsampling presents the best trade-off between accuracy and coverage for macrophyte biomass measurement in the Amazon floodplain.     

Enhanced methane and hydrogen production from municipal solid waste and agro-industrial by-products co-digested with crude glycerol

The effects of crude glycerol on the performance of single-stage anaerobic reactors treating different types of organic waste were examined. A reactor treating the organic fraction of municipal solid waste produced 1400 mL CH₄/d before the addition of glycerol and 2094 mL CH₄/d after the addition of glycerol. An enhanced methane production rate was also observed when a 1:4 mixture of olive mill wastewater and slaughterhouse wastewater was supplemented with crude glycerol. Specifically, by adding 1% v/v crude glycerol to the feed, the methane production rate increased from 479 mL/d to 1210 mL/d. The extra glycerol-COD added to the feed did not have a negative effect on the reactor performance in either case. Supplementation of the feed with crude glycerol also had a significant positive effect on anaerobic fermentation reactors. Hydrogen yield was 26 mmole H₂/g VS added and 15 mmole H₂/g VS added in a reactor treating the organic fraction of municipal solid waste and a 1:4 mixture of olive mill and slaughterhouse wastewater. The addition of crude glycerol to the feed enhanced hydrogen yield at 2.9 mmole H₂/g glycerol added and 0.7 mmole H₂/g glycerol added.     

Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.     

Single-cell protein of Rhodotorula sp. Y-38 from ethanol, acetic acid and acetaldehyde

Summary Continuous cultivation of Rhodotorula sp. Y-38 was carried out on ethanol, acetic acid or acetaldehyde. At a feed concentration of 1.0 % (w/v) ethanol, the cell yield of 64 g/100 g ethanol and crude protein of 52 g/100 g biomass were obtained at D=0.5 h^-1. The respective value of the content of amino acids and nucleic acids was 42.6 and 9.4 g/100 g biomass. At 2.0 % (w/v) acetic acid, cell yield was found to be 50 g/100 g acetic acid at D=0.4 h^-1. The optimum dilution rate ranged between 0.3 and 0.4 h^-1. At 0.05 % (w/v) acetaldehyde, the maximum cell yield was obtained at D=0.14 h^-1.