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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.148, #1-3)

Bundled Slash: A Potential New Biomass Resource for Fuels and Chemicals by Philip H. Steele; Brian K. Mitchell; Jerome E. Cooper; S. Arora (pp. 1-13).

Postharvest residues for southern pine species have not previously been quantified to compare volumes produced from both thinnings and clearcut volumes. A John Deere 1490 Slash Bundler bundled postharvest residues following a first thinning of a 14-year-old stand, a second thinning of a 25-year-old stand, and a clearcut of a naturally regenerated mature stand of 54 years of age. Regardless of stand type, nearly one-fifth of merchantable volume harvested was collected as postharvest residue. Initial bundle moisture contents were 127.3, 81.1, and 49.4% dry basis (db) for the first and second thinnings and mature stands, respectively. Bundle needles content was found to significantly influence the relative moisture contents of the bundles by stand type due to the high moisture content of needles compared to other bundle components. Bundles were stored outside and exposed to very hot and dry conditions and dried very rapidly to lowest moisture contents of 22.8, 14.5, and 13.5% (db) for first and second thinnings and mature stands, respectively. Response to moderating temperatures and higher precipitation resulted in rapid moisture content increase to 69.9, 46.2, and 38.1% (db) for the first and second thinnings and mature stand bundles by the end of the study. Temperature and precipitation and bundle percentage needles content all significantly influenced the rapid moisture content variations observed over the study periods.

Keywords: Harvest; Residues; Bundles; Stand; Slash; Moisture content

Pretreatment Characteristics of Waste Oak Wood by Ammonia Percolation by Jun-Seok Kim; Hyunjoon Kim; Jin-Suk Lee; Joon-Pyo Lee; Soon-Chul Park (pp. 15-22).

A log of waste oak wood collected from a Korean mushroom farm has been tested for ammonia percolation pretreatment. The waste log has different physical characteristics from that of virgin oak wood. The density of the waste wood was 30% lower than that of virgin oak wood. However, there is little difference in the chemical compositions between the woods. Due to the difference in physical characteristics, the optimal pretreatment conditions were also quite different. While for waste oak the optimum temperature was determined to be 130°C, for virgin oak wood the optimum pretreatment was only achieved at 170°C. Presoaking for 12 h with ammonia solution before pretreatment was helpful to increase the delignification efficiency.

Keywords: Waste oak wood; Pretreatment; Ammonia; Enzymatic hydrolysis; Lignin

Pretreatment of Whole-Crop Harvested, Ensiled Maize for Ethanol Production by M. H. Thomsen; J. B. Holm-Nielsen; P. Oleskowicz-Popiel; A. B. Thomsen (pp. 23-33).

To have all-year-round available feedstock, whole-crop maize is harvested premature, when it still contains enough moisture for the anaerobic ensiling process. Silage preparation is a well-known procedure for preserving plant material. At first, this method was applied to obtain high-quality animal feed. However, it was found that such ensiled crops are very suitable for bioenergy production. Maize silage, which consists of hardly degradable lignocellulosic material, hemicellulosic material, and starch, was evaluated for its potential as a feedstock in the production of bioethanol. It was pretreated at low severity (185 °C, 15 min) giving very high glucan (∼100%) and hemicellulose recoveries (<80%)—as well as very high ethanol yield in simultaneous saccharification and fermentation experiments (98% of the theoretical production based on available glucan in the medium). The theoretical ethanol production of maize silage pretreated at 185 °C for 15 min without oxygen or catalyst was 392 kg ethanol per ton of dry maize silage.

Keywords: Maize silage; Bioethanol; Lignocellulose; Pretreatment; Simultaneous saccharification and fermentation

Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading by Tania I. Georgieva; Xiaoru Hou; Troels Hilstrøm; Birgitte K. Ahring (pp. 35-44).

Wheat straw was pretreated by wet explosion using three different oxidizing agents (H₂O₂, O₂, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O₂ as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15–20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.

Keywords: Saccharomyces cerevisiae; Simultaneous saccharification and fermentation; Wet explosion; Pretreatment; Wheat straw; High dry matter; Ethanol

A Comparison between Lime and Alkaline Hydrogen Peroxide Pretreatments of Sugarcane Bagasse for Ethanol Production by Sarita C. Rabelo; Rubens Maciel Filho; Aline C. Costa (pp. 45-58).

Pretreatment procedures of sugarcane bagasse with lime (calcium hydroxide) or alkaline hydrogen peroxide were evaluated and compared. Analyses were performed using 2 × 2 × 2 factorial designs, with pretreatment time, temperature, and lime loading and hydrogen peroxide concentration as factors. The responses evaluated were the yield of total reducing sugars (TRS) and glucose released from pretreated bagasse after enzymatic hydrolysis. Experiments were performed using the bagasse as it comes from an alcohol/sugar factory and bagasse in the size range of 0.248 to 1.397 mm (12–60 mesh). The results show that when hexoses and pentoses are of interest, lime should be the pretreatment agent chosen, as high TRS yields are obtained for nonscreened bagasse using 0.40 g lime/g dry biomass at 70 °C for 36 h. When the product of interest is glucose, the best results were obtained with lime pretreatment of screened bagasse. However, the results for alkaline peroxide and lime pretreatments of nonscreened bagasse are not very different.

Keywords: Lignocellulosic materials; Sugarcane bagasse; Pretreatment; Lime; Hydrogen peroxide; Enzymatic hydrolysis; Statistical analysis

Substrate Dependency and Effect of Xylanase Supplementation on Enzymatic Hydrolysis of Ammonia-Treated Biomass by Rajesh Gupta; Tae Hyun Kim; Yoon Y. Lee (pp. 59-70).

Pretreatment based on aqueous ammonia was investigated under two different modes of operation: soaking in aqueous ammonia and ammonia recycle percolation. These processes were applied to three different feedstocks with varied composition: corn stover, high lignin (HL), and low lignin (LL) hybrid poplars. One of the important features of ammonia-based pretreatment is that most of the hemicellulose is retained after treatment, which simplifies the overall bioconversion process and enhances the conversion efficiency. The pretreatment processes were optimized for these feedstocks, taking carbohydrate retention as well as sugar yield in consideration. The data indicate that hybrid poplar is more difficult to treat than corn stover, thus, requires more severe conditions. On the other hand, hybrid poplar has a beneficial property that it retains most of the hemicellulose after pretreatment. To enhance the digestibility of ammonia-treated poplars, xylanase was supplemented during enzymatic hydrolysis. Because of high retention of hemicellulose in treated hybrid poplar, xylanase supplementation significantly improved xylan as well as glucan digestibility. Of the three feedstocks, best results and highest improvement by xylanase addition was observed with LL hybrid poplar, showing 90% of overall sugar yield.

Keywords: Hybrid poplar; Corn stover; Xylanase; Aqueous ammonia; Pretreatment

Alkali (NaOH) Pretreatment of Switchgrass by Radio Frequency-based Dielectric Heating by Zhenhu Hu; Yifen Wang; Zhiyou Wen (pp. 71-81).

Radio-frequency (RF)-based dielectric heating was used in the alkali (NaOH) pretreatment of switchgrass to enhance its enzymatic digestibility. Due to the unique features of RF heating (i.e., volumetric heat transfer, deep heat penetration of the samples, etc.), switchgrass could be treated on a large scale, high solid content, and uniform temperature profile. At 20% solid content, RF-assisted alkali pretreatment (at 0.1 g NaOH/g biomass loading and 90°C) resulted in a higher xylose yield than the conventional heating pretreatment. The enzymatic hydrolysis of RF-treated solids led to a higher glucose yield than the corresponding value obtained from conventional heating treatment. When the solid content exceeded 25%, conventional heating could not handle this high-solid sample due to the loss of fluidity, poor mixing, and heating transfer of the samples. As a result, there was a significantly lower sugar yield, but the sugar yield of the RF-based pretreatment process was still maintained at high levels. Furthermore, the optimal particle size and alkali loading in the RF pretreatment was determined as 0.25–0.50 mm and 0.25 g NaOH/g biomass, respectively. At alkali loading of 0.20–0.25 g NaOH/g biomass, heating temperature of 90^oC, and solid content of 20%, the glucose, xylose, and total sugar yield from the combined RF pretreatment and the enzymatic hydrolysis were 25.3, 21.2, and 46.5 g/g biomass, respectively.

Keywords: Biomass; Enzymatic hydrolysis; Radio frequency; NaOH pretreatment; Switchgrass

Biological Hydrogen Production Using Chloroform-treated Methanogenic Granules by Bo Hu; Shulin Chen (pp. 83-95).

In fermentative hydrogen production, the low-hydrogen-producing bacteria retention rate limits the suspended growth reactor productivity because of the long hydraulic retention time (HRT) required to maintain adequate bacteria population. Traditional bacteria immobilization methods such as calcium alginate entrapment have many application limitations in hydrogen fermentation, including limited duration time, bacteria leakage, cost, and so on. The use of chloroform-treated anaerobic granular sludge as immobilized hydrogen-producing bacteria in an immobilized hydrogen culture may be able to overcome the limitations of traditional immobilization methods. This paper reports the findings on the performance of fed-batch cultures and continuous cultures inoculated with chloroform-treated granules. The chloroform-treated granules were able to be reused over four fed-batch cultures, with pH adjustment. The upflow reactor packed with chloroform-treated granules was studied, and the HRT of the upflow reactor was found to be as low as 4 h without any decrease in hydrogen production yield. Initial pH and glucose concentration of the culture medium significantly influenced the performance of the reactor. The optimum initial pH of the culture medium was neutral, and the optimum glucose concentration of the culture medium was below 20 g chemical oxygen demand/L at HRT 4 h. This study also investigated the possibility of integrating immobilized hydrogen fermentation using chloroform-treated granules with immobilized methane production using untreated granular sludge. The results showed that the integrated batch cultures produced 1.01 mol hydrogen and 2 mol methane per mol glucose. Treating the methanogenic granules with chloroform and then using the treated granules as immobilized hydrogen-producing sludge demonstrated advantages over other immobilization methods because the treated granules provide hydrogen-producing bacteria with a protective niche, a long duration of an active culture, and excellent settling velocity. This integrated two-stage design for immobilized hydrogen fermentation and methane production offers a promising approach for modifying current anaerobic wastewater treatment processes to harvest hydrogen from the existing systems.

Keywords: Biological hydrogen production; Chloroform treatment; Granular; Immobilization; Integration with methane production

Effect of Furfural, Vanillin and Syringaldehyde on Candida guilliermondii Growth and Xylitol Biosynthesis by Christine Kelly; Opal Jones; Christopher Barnhart; Curtis Lajoie (pp. 97-108).

Xylitol is a five-carbon sugar alcohol with established commercial use as an alternative sweetener and can be produced from hemicellulose hydrolysate. However, there are difficulties with microbiological growth and xylitol biosynthesis on hydrolysate because of the inhibitors formed from hydrolysis of hemicellulose. This research focused on the effect of furfural, vanillin, and syringaldehyde on growth of Candida guilliermondii and xylitol accumulation from xylose in a semi-synthetic medium in microwell plate and bioreactor cultivations. All three compounds reduced specific growth rate, increased lag time, and reduced xylitol production rate. In general, increasing concentration of inhibitor increased the severity of inhibition, except in the case of 0.5 g vanillin per liter, which resulted in a faster late batch phase growth rate and increased biomass yield. At concentrations of 1 g/l or higher, furfural was the least inhibitory to growth, followed by syringaldehyde. Vanillin most severely reduced specific growth rate. All three inhibitors reduced xylitol production rate approximately to the same degree.

Keywords: Xylitol; Inhibitor; Furfural; Vanillin; Syringaldehyde; Toxicity; Hemicellulose hydrolysate

Production and Characterization of Biodiesel from Tung Oil by Ji-Yeon Park; Deog-Keun Kim; Zhong-Ming Wang; Pengmei Lu; Soon-Chul Park; Jin-Suk Lee (pp. 109-117).

The feasibility of biodiesel production from tung oil was investigated. The esterification reaction of the free fatty acids of tung oil was performed using Amberlyst-15. Optimal molar ratio of methanol to oil was determined to be 7.5:1, and Amberlyst-15 was 20.8wt% of oil by response surface methodology. Under these reaction conditions, the acid value of tung oil was reduced to 0.72mg KOH/g. In the range of the molar equivalents of methanol to oil under 5, the esterification was strongly affected by the amount of methanol but not the catalyst. When the molar ratio of methanol to oil was 4.1:1 and Amberlyst-15 was 29.8wt% of the oil, the acid value decreased to 0.85mg KOH/g. After the transesterification reaction of pretreated tung oil, the purity of tung biodiesel was 90.2wt%. The high viscosity of crude tung oil decreased to 9.8mm²/s at 40 °C. Because of the presence of eleostearic acid, which is a main component of tung oil, the oxidation stability as determined by the Rancimat method was very low, 0.5h, but the cold filter plugging point, −11 °C, was good. The distillation process did not improve the fatty acid methyl ester content and the viscosity.

Keywords: Biodiesel; Eleostearic acid; Esterification by Amberlyst-15; Fuel properties; Response surface methodology; Tung oil

Yeast Biomass Production in Brewery’s Spent Grains Hemicellulosic Hydrolyzate by Luís C. Duarte; Florbela Carvalheiro; Sónia Lopes; Inês Neves; Francisco M. Gírio (pp. 119-129).

Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery’s spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h⁻¹, 0.61 g g⁻¹, and 0.56 g l⁻¹ h⁻¹, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.

Keywords: Debaryomyces hansenii ; Biomass; Single-cell protein; Hemicellulosic hydrolyzate; Brewery’s spent grains; Agro-industrial residues upgrading; Yeast extract

Lipase-Catalyzed Transesterification of Rapeseed Oil for Biodiesel Production with tert-Butanol by Gwi-Taek Jeong; Don-Hee Park (pp. 131-139).

Biodiesel is a fatty acid alkyl ester that can be derived from any vegetable oil or animal fat via the process of transesterification. It is a renewable, biodegradable, and nontoxic fuel. In this paper, we have evaluated the efficacy of a transesterification process for rapeseed oil with methanol in the presence of an enzyme and tert-butanol, which is added to ameliorate the negative effects associated with excess methanol. The application of Novozym 435 was determined to catalyze the transesterification process, and a conversion of 76.1% was achieved under selected conditions (reaction temperature 40 °C, methanol/oil molar ratio 3:1, 5% (w/w) Novozym 435 based on the oil weight, water content 1% (w/w), and reaction time of 24h). It has also been determined that rapeseed oil can be converted to fatty acid methyl ester using this system, and the results of this study contribute to the body of basic data relevant to the development of continuous enzymatic processes.

Keywords: Biodiesel; Novozym 435; tert-Butanol; Methanolysis

Bioethanol Production Optimization: A Thermodynamic Analysis by Víctor H. Álvarez; Elmer Ccopa Rivera; Aline C. Costa; Rubens Maciel Filho; Maria Regina Wolf Maciel; Martín Aznar (pp. 141-149).

In this work, the phase equilibrium of binary mixtures for bioethanol production by continuous extractive process was studied. The process is composed of four interlinked units: fermentor, centrifuge, cell treatment unit, and flash vessel (ethanol-congener separation unit). A proposal for modeling the vapor–liquid equilibrium in binary mixtures found in the flash vessel has been considered. This approach uses the Predictive Soave–Redlich–Kwong equation of state, with original and modified molecular parameters. The congeners considered were acetic acid, acetaldehyde, furfural, methanol, and 1-pentanol. The results show that the introduction of new molecular parameters r and q in the UNIFAC model gives more accurate predictions for the concentration of the congener in the gas phase for binary and ternary systems.

Keywords: Bioreactors; Fermentation; Phase equilibria; Predictive Soave–Redlich–Kwong

Oxidation in Acidic Medium of Lignins from Agricultural Residues by Gisele Aparecida Amaral Labat; Adilson Roberto Gonçalves (pp. 151-161).

Agricultural residues as sugarcane straw and bagasse are burned in boilers for generation of energy in sugar and alcohol industries. However, excess of those by-products could be used to obtain products with higher value. Pulping process generates cellulosic pulps and lignin. The lignin could be oxidized and applied in effluent treatments for heavy metal removal. Oxidized lignin presents very strong chelating properties. Lignins from sugarcane straw and bagasse were obtained by ethanol–water pulping. Oxidation of lignins was carried out using acetic acid and Co/Mn/Br catalytical system at 50, 80, and 115 °C for 5 h. Kinetics of the reaction was accomplished by measuring the UV-visible region. Activation energy was calculated for lignins from sugarcane straw and bagasse (34.2 and 23.4 kJ mol⁻¹, respectively). The first value indicates higher cross-linked formation. Fourier-transformed infrared spectroscopy data of samples collected during oxidation are very similar. Principal component analysis applied to spectra shows only slight structure modifications in lignins after oxidation reaction.

Keywords: Sugarcane bagasse; Sugarcane straw; Oxidation in acidic medium; Chelating agents; FTIR; PCA

Kinetic Modeling and Parameter Estimation in a Tower Bioreactor for Bioethanol Production by Elmer Ccopa Rivera; Aline Carvalho da Costa; Betânia Hoss Lunelli; Maria Regina Wolf Maciel; Rubens Maciel Filho (pp. 163-173).

In this work, a systematic method to support the building of bioprocess models through the use of different optimization techniques is presented. The method was applied to a tower bioreactor for bioethanol production with immobilized cells of Saccharomyces cerevisiae. Specifically, a step-by-step procedure to the estimation problem is proposed. As the first step, the potential of global searching of real-coded genetic algorithm (RGA) was applied for simultaneous estimation of the parameters. Subsequently, the most significant parameters were identified using the Placket–Burman (PB) design. Finally, the quasi-Newton algorithm (QN) was used for optimization of the most significant parameters, near the global optimum region, as the initial values were already determined by the RGA global-searching algorithm. The results have shown that the performance of the estimation procedure applied in a deterministic detailed model to describe the experimental data is improved using the proposed method (RGA–PB–QN) in comparison with a model whose parameters were only optimized by RGA.

Keywords: Ethanol fermentation; Parameter estimation; Modeling; Optimization techniques; Artificial intelligence

Analysis of Kinetic and Operational Parameters in a Structured Model for Acrylic Acid Production through Experimental Design by B. H. Lunelli; E. C. Rivera; E. C. Vasco de Toledo; M. R. Wolf Maciel; R. Maciel Filho (pp. 175-187).

In biotechnological processes, a great number of factors can influence the income productivity and conversion. Normally, it is not evident which of these factors are the most important and how they interact. In this work, multivariate analysis techniques are used as experimental design coupled to a detailed deterministic model to identify the parameters with the most significant impact on the model to represent well the acrylic acid production process. It is proposed as an alternative process, having sugarcane as feedstock, to the petrochemical-based ones that have significant environmental impacts for their production. To increase the competitiveness of renewable acrylic-acid-based process, it is necessary to find out working conditions near the optimal region, which is not an easy task, as the process is multivariable and non-linear. The mapping of the dynamics of the developed process is made using techniques of factorial design together with the methodology of Plackett–Burman. It is shown that it is possible to increase the process performance by choosing optimized conditions for the reactor operation.

Keywords: Experimental design; Plackett–Burman design; Factorial design; Biotechnological process; Acrylic acid; Structured model

Optimization of Oligosaccharide Synthesis from Cellobiose by Dextransucrase by Misook Kim; Donal F. Day (pp. 189-198).

There is a growing market for oligosaccharides as sweeteners, prebiotics, anticariogenic compounds, and immunostimulating agents in both food and pharmaceutical industries. Interest in novel carbohydrate-based products has grown because of their reduced toxicity and low immune response. Cellobiose is potentially valuable as a nondigestible sugar. The reaction of cellobiose, as an acceptor with a sucrose as a donor, catalyzed by a dextransucrase from Leuconostoc mesenteroides B-512FMCM, produced a series of cellobio-oligosaccharides. This production system was optimized using a Box–Behnken experimental design for 289 mM of sucrose and 250 mM of cellobiose and 54 U of the enzyme at pH 5.2 and 30 °C, to produce maximum yields of oligosaccharide.

Keywords: Oligosaccharide; Cellobiose; Dextransucrase; Leuconostoc mesenteroides B-512FMCM; Box-Behnken experimental design

Fermentation Kinetics for Xylitol Production by a Pichia stipitis d-Xylulokinase Mutant Previously Grown in Spent Sulfite Liquor by Rita C. L. B. Rodrigues; Chenfeng Lu; Bernice Lin; Thomas W. Jeffries (pp. 199-209).

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3Δ) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h⁻¹). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g_xylose/g_cel h) and xylitol production (0.059 g_xylitol/g_cel h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

Keywords: Xylitol; Yeast; Xylose; Ammonia spent sulfite liquor-SSL; Inoculum adaptation; Enzymes

Selective Enrichment of a Methanol-Utilizing Consortium Using Pulp and Paper Mill Waste Streams by Gregory R. Mockos; William A. Smith; Frank J. Loge; David N. Thompson (pp. 211-226).

Efficient utilization of carbon inputs is critical to the economic viability of the current forest products sector. Input carbon losses occur in various locations within a pulp mill, including losses as volatile organics and wastewater. Opportunities exist to capture this carbon in the form of value-added products such as biodegradable polymers. Waste-activated sludge from a pulp mill wastewater facility was enriched for 80 days for a methanol-utilizing consortium with the goal of using this consortium to produce biopolymers from methanol-rich pulp mill waste streams. Five enrichment conditions were utilized: three high-methanol streams from the kraft mill foul condensate system, one methanol-amended stream from the mill wastewater plant, and one methanol-only enrichment. Enrichment reactors were operated aerobically in sequencing batch mode at neutral pH and 25°C with a hydraulic residence time and a solids retention time of 4 days. Non-enriched waste activated sludge did not consume methanol or reduce chemical oxygen demand. With enrichment, however, the chemical oxygen demand reduction over 24-h feed/decant cycles ranged from 79 to 89%, and methanol concentrations dropped below method detection limits. Neither the non-enriched waste-activated sludge nor any of the enrichment cultures accumulated polyhydroxyalkanoates (PHAs) under conditions of nitrogen sufficiency. Similarly, the non-enriched waste activated sludge did not accumulate PHAs under nitrogen-limited conditions. By contrast, enriched cultures accumulated PHAs to nearly 14% on a dry weight basis under nitrogen-limited conditions. This indicates that selectively enriched pulp mill waste activated sludge can serve as an inoculum for PHA production from methanol-rich pulp mill effluents.

Keywords: Foul condensate; Waste-activated sludge; Polyhydroxyalkanoates; Pulp mill; Natural fiber reinforced thermoplastic composite

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol by Álvaro Daniel Teles Pinheiro; Maria Valderez Ponte Rocha; Gorete R. Macedo; Luciana R. B. Gonçalves (pp. 227-234).

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.

Keywords: Ethanol; Cashew apple juice; Saccharomyces cerevisiae ; Batch cultivation; Kinetic parameters

Atmospheric Pressure Liquefaction of Dried Distillers Grains (DDG) and Making Polyurethane Foams from Liquefied DDG by Fei Yu; Zhiping Le; Paul Chen; Yuhuan Liu; Xiangyang Lin; Roger Ruan (pp. 235-243).

In this study, dried distillers grains (DDG) was liquefied in acidic conditions at atmospheric pressure, and polyurethane foams were subsequently prepared from the liquefied DDG. Liquefaction was examined over a range of conditions including liquefaction time of 1–3 h, temperature of 150–170 °C, sulfuric acid (as catalyst) concentration of 1.0–3.0 wt%, and liquefaction solvent (ethylene carbonate) to DDG ratio of 3:1–5:1. The bio-polyols in the liquefied DDG were rich in hydroxyl groups, which can react with methylene diphenyl diisocyanate (MDI) to form cross-linked polyurethane networks. The biodegradability of the prepared polyurethane foams was also evaluated. This study strives to broaden the application of DDG as a feedstock for bio-polyurethane preparation.

Keywords: Dried distillers grains; Liquefaction; Bio-polyols; Polyurethane; Biodegradable

Bacterial Cellulose Production by Acetobacter xylinum Strains from Agricultural Waste Products by Sasithorn Kongruang (pp. 245-256).

Bacterial cellulose is a biopolysaccharide produced from the bacteria, Acetobacter xylinum. Static batch fermentations for bacterial cellulose production were studied in coconut and pineapple juices under 30 °C in 5-l fermenters by using three Acetobacter strains: A. xylinum TISTR 998, A. xylinum TISTR 975, and A. xylinum TISTR 893. Experiments were carried out to compare bacterial cellulose yields along with growth kinetic analysis. Results showed that A. xylinum TISTR 998 produced a bacterial cellulose yield of 553.33 g/l, while A. xylinum TISTR 893 produced 453.33 g/l and A. xylinum TISTR 975 produced 243.33 g/l. In pineapple juice, the yields for A. xylinum TISTR 893, 975, and 998 were 576.66, 546.66, and 520 g/l, respectively. The strain TISTR 998 showed the highest productivity when using coconut juice. Morphological properties of cellulose pellicles, in terms of texture and color, were also measured, and the textures were not significantly different among treatments.

Keywords: Bacterial cellulose; Acetobacter xylinum ; Texture; Coconut juice; Pineapple

Overview of Special Session B—Compositional and Structural Analysis of Biomass by Bonnie Hames (pp. 257-260).

Special Session B at the 29th Symposium on Biotechnology for Fuels and Chemicals was the first invited session at this symposium devoted to analytical methods. The special topic was added in response to numerous requests for information on new and innovative methods that could be applied in the growing renewable fuels industry. Presentation topics include analytical methods for the characterization and analysis of maize traits, tools for investigating cell wall limitations to enzymatic degradation, methods for customizing enzyme cocktails for biomass, new techniques for the analysis of carbohydrates, analytical methods that enhance our understanding of pretreatment, improved methods for monitoring process intermediates, and published standard analytical methods for biomass conversion processes.

Keywords: Analytical chemistry; Enzymes; Chromatography; Standard methods; Feedstock; Biomass; Forage; Carbohydrates; Imaging; Pretreatment; Saccharification; Process monitoring

What can be Learned from Silage Breeding Programs? by Aaron J. Lorenz; James G. Coors (pp. 261-270).

Improving the quality of cellulosic ethanol feedstocks through breeding and genetic manipulation could significantly impact the economics of this industry. Attaining this will require comprehensive and rapid characterization of large numbers of samples. There are many similarities between improving corn silage quality for dairy production and improving feedstock quality for cellulosic ethanol. It was our objective to provide insight into what is needed for genetic improvement of cellulosic feedstocks by reviewing the development and operation of a corn silage breeding program. We discuss the evolving definition of silage quality and relate what we have learned about silage quality to what is needed for measuring and improving feedstock quality. In addition, repeatability estimates of corn stover traits are reported for a set of hybrids. Repeatability of theoretical ethanol potential measured by near-infrared spectroscopy is high, suggesting that this trait may be easily improved through breeding. Just as cell wall digestibility has been factored into the latest measurements of silage quality, conversion efficiency should be standardized and included in indices of feedstock quality to maximize overall, economical energy availability.

Keywords: Silage breeding; Corn stover; Repeatability; Quality

Permethylation Linkage Analysis Techniques for Residual Carbohydrates by Neil P. J. Price (pp. 271-276).

Permethylation analysis is the classic approach to establishing the position of glycosidic linkages between sugar residues. Typically, the carbohydrate is derivatized to form acid-stable methyl ethers, hydrolyzed, peracetylated, and analyzed by gas chromatography-mass spectrometry. The position of glycosidic linkages in the starting carbohydrate are apparent from the mass spectra as determined by the location of acetyl residues. The completeness of permethylation is dependent upon the choice of base catalyst and is readily confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry mass spectrometry. For the permethylation of β-cyclodextrin, Hakomori dimsyl base is shown to be superior to the NaOH–dimethyl sulfoxide system, and the use of the latter resulted in selective under-methylation of the 3-hydroxy groups. These techniques are highly applicable to residual carbohydrates from biofuel processes.

Keywords: Carbohydrate; Linkage analysis; Permethylation; Mass spectrometry

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.148, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.148, #1-3)