13.2.1 Supercritical Fluid Processing

Samples of Sitka spruce (Picea sitchensis) and Alaska birch (Betula neoalaskana) were dried to a moisture content of 4 wt% and processed in a Willey mill to pass through a 2 mm sieve. Approximately 4.5 g of each biomass stream were intro­duced into a 75 ml high-pressure reactor (Parr Instruments model 4740), where methanol (35 ml) was introduced to a volume ratio of 0.46 [18]. The reactor was placed inside a temperature-controlled heating block, where the internal reaction temperature was raised to 375°C. Both temperature and pressure were monitored with reaction time by means of a thermocouple and a pressure gauge mounted directly to the reactor vessel. Given the batch nature of the reactor, the pressure increased to above the supercritical pressure point of methanol (8.1 MPa) and increased to a maximum of 42 MPa. At this maximum pressure, the reactor was removed from the heater block and quenched in an ice-water bath until cold. The solid material and liquid were separated by means of vacuum filtration through a glass fiber mat, creating a bio-oil fraction and an insoluble biochar component and yields determined.

13.2.2 Chemical Characterization

Fourier transform infrared spectra were obtained on wood and biochar samples in the attenuated total reflectance (ATR) mode (ZnSe, single bounce) on an Avatar 370 spectrometer (ThermoNicolet). The collected spectra were ATR and baseline corrected using Omnic v7 software. Lignin condensation indices (CIs) were cal­culated according to Faix [29] with a slight modification to the method. The sum of all spectra minima intensity between 1,500 and 1,050 cm-1 was divided by the sum of all spectra maxima intensity between 1,600 and 1,030 cm-1 and was used to calculate the CI.

The volatile components in the bio-oil (methanol soluble fraction) were determined by GC-MS analysis on a Polaris Q instrument (ThermoQuest). An aliquot of the methanol soluble fraction (200 il) was transferred to a GC vial to which dichloromethane (1 ml containing 0.1 mg/ml anthracene as an internal standard) was added. Separation was achieved using a ZB-1 capillary column (Phenomenex, 30 m, 0.25 mm 0) with helium as a carrier gas and a temperature program of 40 (2 min) to 300°C (10 min) at the ramp rate of 5°C/min. Data analysis was performed using the Xcalibur software v2 (Thermoscientific). The compounds were identified by comparison with standards, their corresponding mass spectra and NIST 2008 library.

Reactor configuration and its operation mode are the main parameters that can affect the overall hydrogen production in dark fermentative systems. Reactor configuration is very important for microenvironment, microbial population, hydrodynamic behavior, etc. To date many of the laboratory scale dark fermen­tative experiments were in batch mode because it can be easily operated and flexible. But for industrial applications, it is important to operate the reactor continuously to keep continuous usage of the industrial waste and wastewater in accordance with continuous hydrogen production. Many of the studies were done by using continuous stirred tank reactor (CSTR). Suspended cell culture systems are advantageous because of the good mass transfer between microorganisms and substrates. But the disadvantage is the wash-out of cells at low hydraulic retention time conditions. Immobilization is an important technique to improve the system performance for continuous systems. Immobilization techniques can generally be divided into three main categories; adsorption (biofilm formation), encapsulation and entrapment. It is important to select an economical and durable method. Every immobilization technique and each material have their own advantages and disadvantages [89]. It is shown that immobilization can increase the system per­formance of dark fermentative hydrogen production [90]. For different immobi­lization techniques (attachment, granulation, flocculation and entrapment) ranging from 0.93 to 7.33 l/l/h at low HRT values between 0.5 h and 4 h. is achieved where suspended systems give hydrogen production rates of 0.15-0.58 l/l/h at 6 h HRT, demonstrating that immobilization of microorganisms can greatly increase reactor performance and biomass retention [91, 92].

12.3.2.1 Vanillin Market

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is widely used as flavoring and fragrance ingredient in food, cosmetic and as intermediate for the synthesis of several second generation fine chemicals (as veratraldehyde, protocatechualde — hyde, and respective acids) and pharmaceuticals (as papaverine, levodopa and cyclovalone) [91, 92].

The global market for vanillin and ethyl vanillin is estimated as high as 16 thousand t/year, with 2 thousand t coming from lignin-based vanillin. Production of pure natural vanillin is estimated around 40 t/year [93].Vanillin market is mainly constituted by large multinational holders in the field of flavor and fra­grance, chocolate and ice cream production, and synthesis of pharmaceuticals.

^ °н он он OH OH oh oh O о? H О <?H

O О^н^бс^, О O HO O

— r ‘-^:

internal standard

LU

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00

Time (min)

Fig. 12.5 Gas chromatogram with mass selective detector of monomeric products obtained from catalyzed hydrothermal degradation of organosolv beech lignin [69]. Courtesy of Dr. Detlef Schmiedl, Fraunhofer Institute for Chemical Technology, Germany

Today there are two commercial types of vanillin: (1) synthetic vanillin, derived from petrochemical guaiacol and glyoxylic acid or lignosulfonates and (2) vanilla extract obtained from the cured beans, or pods, of tropical Vanilla orchids [94, 95]. The raw material costs turn the natural vanillin more expensive than the synthetic counterpart [94]. Hence, synthetic vanillin became competitive and widely used.

There are only few significant manufacturers of vanillin in the world. Rhodia SA dominates the market producing vanillin by the cathecol-guaiacol route. Borregaard (Norway) is the second largest vanillin producer and the only current producer by oxidation of lignosulfonates. Despite the advantages of the cathecol — guaiacol route over alternatives, this process is dependent of petroleum-derived compounds, in opposition with the process by lignin oxidation.

The microbes are single-celled organisms that multiply by simple cell division and derive energy for growth and cell functioning by oxidizing iron and sulfur. Oxi­dation involves the removal of electrons from a substance. In biomining process, the microbes remove electrons from dissolved iron (ferrous iron) converting it to another form of iron (ferric iron); electrons are removed from sulfur converting it to sulfuric acid. They obtain carbon for their cellular bodies from carbon dioxide (CO2) in the atmosphere and also require a sulfuric acid environment to grow. This acidic environment is helpful in growth of these microorganisms but acidity must be less than pH 2.5, which is more acidic than vinegar.

The biomining microorganisms do not cause diseases in humans, animals, or plants. Because their food source is inorganic (sulfur and iron) and because they must live in a sulfuric acid environment, they cannot survive in or on plants and animals. These microbes are conveniently grouped within temperature ranges at which they grow and where they are found in the natural environment:

• Ambient temperature bacteria (mesophiles)

• Moderately-thermophilic (heat-loving) bacteria

• Extremely-thermophilic (heat-loving) bacteria

Ambient temperature bacteria (mesophiles). These cylindrical-shaped biomining bacteria are about 1 im long and V2 im in diameter (1 im is 4/100,000 of an inch). About 1,500 of these bacteria could lay end-to-end across a pin head. They only grow and function from 10 to 40°C (50 to 104°F). If the temperature is too low, these bacteria become dormant. If the temperature exceeds 45°C (113°F), the organisms die as their proteins coagulate similar to cooking an egg. Acidithio — bacillus ferrooxidans belong to this group of bacteria. Others include Leptospir — illum ferrooxidans and species of Ferroplasma.

Moderately-thermophilic (heat-loving) bacteria. These bacteria are similar to the “mesophilic” biomining bacteria, except they are somewhat larger in length — about 2-5 im long and they only grow and perform when the temperature exceeds 40°C (104°F). The moderate thermophiles die when the temperature exceeds 60°C (140°F). Examples of moderate thermophiles are species of Sulfobacillus and Acidithiobacillus caldus.

Extremely-thermophilic Archaea. While similar in size (one micrometer in diameter) to ambient temperature bacteria, Archaea have a different molecular organization. In the tree of life, Archaea occupy the lowest branch and are extant members of an offshoot of primitive microbes. They have a spherical shape and characteristically lack a rigid cell wall, rather the contents of the single cell are enclosed by a membrane. These microbes, nevertheless, are extremely robust growing and performing only at temperatures between 60 (140°F) and 85°C (185°F). Examples of extremely-thermophilic Archaea used in biomining are Acidianus brierleyi, Sulfolobus metallicus and Metallosphaera sedula [8].

A process that employs ethanol fractionation as a pretreatment approach to separate cellulose, hemicelluloses, lignin and extractives from woody biomass has been proposed by Lignol Innovation (Fig. 11.1) [42]. The obtained cellulose fraction is claimed to be highly susceptible to enzymatic hydrolysis, and the generated glucose of a high yield is readily converted into ethanol, or possibly used as sugar platform chemicals via saccharification and fermentation. In addi­tion, the liquor rich in lignin, furfural, xylose, acetic acid and lipophylic extrac­tives, can be separated by well-established unit operations. The ethanol is recovered and recycled back in the whole process. The recycled process water is of high quality, low BOD5 and suitable for the overall system process closure. The proposed steps for product separation are shown as follows: (1) After the cooking, the cooking liquor is turned into a black liquor, which is further subjected to

Cellulose

I

Saccharification

and

Fermentation

precipitation to recover lignin by diluting the black liquor with enormous process steam and filtering, washing and drying the precipitated lignin. (2) The ethanol in the black liquor is recovered and recycled by flashing the black liquor and com­pensating the vapors. With respect to the filtrate and washing liquor, they are distilled to achieve a higher concentration. (3) Acetic acid, furfural, xylose and extractives are separated from the distillation column. (4) Oligosaccharides are converted into sugars for fermentation to produce more ethanol using mild acid hydrolysis. Based on the economic evaluation, it has been claimed that this process can be operated in a plant as a small scale as 100 mt per day.

Ethanol fractionation process in combination with ultra-filtration has been designed by Garcia et al. [43]. The main unit operations are cooking, flash oper­ation, washing stage and ultra-filtration. The cooking operation is conducted in a pressurized reactor. Flash operation is used to recover stream mixtures of ethanol and water. In the washing stage, the obtained fibers are washed with mixtures of ethanol and water under the same concentration of the cooking liquor. The lignin dissolved in the black liquor is separated into homogeneous fractions by using ultra-filtration and then is subjected to precipitation with water. To achieve fully solvent recycle, liquor faction after lignin precipitation is sent to distillation unit to recover the ethanol/water mixtures, whereas the residue composed of water and co-products is treated by heating in a flash unit to recover a clean water stream for lignin precipitation. By using the simulation software Aspen Plus, the energetic and economical efficiencies of the ethanol fractionation are evaluated considering several units, including reaction, solid fraction washing, products recovery and liquid fraction processing. Mass and energy balances are evaluated in terms of yield, solvents/reactants recovery and energy consumption. In addition, pinch technology has been applied to improve the heat exchange network of the ethanol fractionation process reducing the associated utilities requirements, making the process more competitive as compared to the soda process.

In a recently proposed ethanol extraction process, a pre-hydrolysis step is applied to remove the hemicelluloses of wood chips [44]. The open diagram in Fig. 11.2 shows the steps required for recovering the hemicelluloses from the pre­hydrolysis liquor (PHL) in a separate stream. In the pre-hydrolysis step, hemicelluloses are extracted accompanying removal of a part of lignin.

Subsequently, the dissolved lignin is precipitated by decreasing the pH of the PHL to 2 using sulfuric acid. Then the precipitated lignin is subjected to a filter washer. The recovery of hemicelluloses is conducted by the addition of ethanol into the acidified PHL and further separated from the ethanol/water solution in a filter washer. The pre-hydrolyzed feedstock, with increased porosity, is subjected to the ethanol fractionation for removing the remaining lignin, similar to the ethanol pulping process. The dissolved lignin in the ethanol extraction step is recovered by acidification and then separated in a filter washer. After the ethanol extraction, cellulose is remained as a solid residue associated with a small amount of lignin, which can be further removed in an elemental chlorine free (ECF) based bleaching process.

Separation of lignin from spent liquor is generally based on the lignin insolu­bility in acid water. The recovery of lignin in an acid process consists of the following stages: precipitation of the lignin fraction with higher molecular weight; separation of the precipitate by decantation, thickening, centrifugation or filtration; washing with water to reduce impurities; further thickening to remove the water retained in the washing stage; drying of lignin. However, lignin dissolved in alkaline ethanol liquor is difficult to precipitate because the process decreasing the spent liquor pH to around 2 requires a large amount of acid to neutralize.

Generally, there are two typical methods to recover lignin from acidic ethanol — soluble liquor. One is dilution of spent liquors in water directly [20, 45], which is characterized by low speeds and sometimes difficult to filtrate or centrifuge due to the generation of a rather stable colloidal suspension. The other way consists of recovery of the alcohol from spent liquor in recovery tower under reduced pres­sure, then precipitation of lignin in water [46]. This procedure is usually ineffective and difficult to control, because lignin tends to precipitate as a sticky tar in the internal surfaces of the recovery tower, reducing the recovery of alcohol. The modified method is the evaporation of 60-65% alcohol in a flash tank, cooling the spent liquor to a temperature above 70°C (to avoid the precipitation) and diluting by injection of the liquor into water through a Venturi tube [47].

In a recent study, two feasible laboratory-scale ways are proposed to recover lignin by precipitation [48]. The laboratory-scale representation of a system involving the reduction of ethanol concentration in the spent liquors by evapora­tion in a flash tank to 30% (v/v), dilution ratio of 1:1, at 40°C and centrifugation, appeared as the best alternative for lignin recovery (45% of precipitate with a purity of 94%, yielding 42% pure lignin). Another feasible procedure involved lignin precipitation and recovery from the spent liquors by dilution with water under a dilution ratio of 1:2. This method yielded 41% pure lignin, yet from a precipitate of 48% with 87% purity (much more contaminated, mainly with car­bohydrates). The temperature of the treatments affects the recovery process. In both cases, the most suitable dilution conditions are at room temperature or 40°C.

In addition, ultra-filtration membrane allows to recover lignin with specific molecular weight, but the cost is relatively high [49, 50]. For instance, ultra­filtration has been used to fractionate the lignin dissolved in ethanol-soluble liquor. The ultra-filtration module is a pilot unit equipped with a stainless steel tank with water jacket for temperature control, a recirculation pump and a set of tubular ceramic membranes of different cut-offs in the interval 5-15 kDa [51]. Four different cutoff fractions are obtained: less than 5 kDa fraction; 5-10 kDa fraction; 10-15 kDa fraction and more than 15 kDa fraction. After the ultra-filtration, the obtained lignin has a relatively homogeneous molecular weight distribution.

The most frequent catalysts used in lignin oxidation with O2 in alkaline medium are transition metal salts, such as CuO, CuSO4, FeCl3, and Fe2O3, which have high oxidation potential and easily would allow electron transference from the aromatic rings of lignin; at the same time this high oxidation potential turns the regeneration of the metal salt in the catalytic cycle more difficult. The oxidation with catalyst has been extensively tested on model compounds of lignin, most of them mono­mers. A recent and comprehensive review was recently published by Zakzeski et al. [90] about oxidative catalysis and other perspectives of the catalytic valo­rization of lignin.

Oxidations experiments were performed by Mathias and Rodrigues [118] using CuSO4 (4% of lignin weight) and comparing the reaction rate and yields to the non-catalyzed reaction of kraft lignin at the same conditions. The yield on vanillin was similar, as well as the time to maximum. However, a low degradation rate was found in the case of catalyzed reaction. The same salt was tested in the oxidation of lignosulfonates used at 20% of lignin weight [113]. The authors reported an increment of 1.3 and 1.4 of the yields of vanillin and syringaldehyde produced in the non-catalyzed reaction.

Tarabanko et al., reported 12-13%wt (lignin basis) of vanillin in batch oxida­tion of lignosulfonates using about 16 g/l of Cu(OH)2 [139]. The yield of non-catalyzed reaction was 5.5 wt. % on vanillin at 40 min. The lignosulfonate liquor of the same origin was the raw material for further experiences in contin­uous process [159]. In this case, the authors reported also the syringaldehyde yield. Copper wire and cupric oxide wire were tested as catalyst and simultaneously as reactor packing. In this work, the conclusions about the catalyst effect are some­what difficult due to the simultaneous variation of parameters as, for example, the oxygen rate in continuous process. However, for lignin from other origin, the comparison with non-catalyzed reaction leads us to notice an increase of aldehydes yield (from 1.2-1.8 wt% to 1.5-3.0 wt% on lignin bases) [159].

Other combinations of catalysts were tested in the oxidation of an hardwood lignin: the mixture of CuSO4 with FeCl3 and CuO with Fe2O3 [110]. At reaction temperature of 433 K the yields were incremented by CuSO4/FeCl3 (4.5% for vanillin and 7.4% for syringaldehyde) in comparison with the non-catalyzed reaction at similar reaction conditions (2.5% for vanillin and 4.5% for syringal — dehyde). Based on data reported for 443 K, the additional effect of FeCl3 was evidenced: 4.7 and 9.5% of vanillin and syringaldehyde, respectively, against 3.5 and 6.5% for the reaction at similar conditions using CuSO4 alone.

Besides the Cu(II) catalysis, Bj0rsvik and Minisci [155] also tested salts of Co(II) and Ce(IV) salts in lignin oxidation. The efficiency of the Cu(II) and Co(II) catalysts was similar: 5.9 and 5.8%, respectively; however, in the reaction cata­lyzed by Cu(II), the maximum yield of vanillin was reached faster (70 min) than with Co(II) (90 min). The salt of Ce(IV) shows a lower efficiency in the oxidation of lignosulfonate, which was ascribed to the higher difficulty to reoxidation of the

Ce(III). Besides Cu and Co salts, two commercial platinum-alumina catalysts were studied by Villar et al. [123]. The copper salts produced better results; however, with similar yield of that obtained without catalyst. Moreover, cobalt and plati­num-alumina catalysts showed a negative effect on lignin conversion.

Sales et al. [111, 112] used palladium catalyst supported on y-alumina for oxidation of sugarcane bagasse lignin (batch and continuous). The catalyst revealed effective on increasing the rate of formation of aldehydes, 10-20 times higher compared to the correspondent non-catalytic reactions.

A heterogeneous catalyst was recently reported in literature [124] as effective in the production of aldehydes: the perovskite-type oxide LaFe1_xCuxO3 (x = 0, 0.1, 0.2). The maximum yield of p-hydroxybenzaldehyde, vanillin, and syringaldehyde was significantly improved with the catalyst LaFe0.8Cu0.2O3: 2.49% (at 120 min), 4.56% (at 60 min) and 11.51% (at 30 min), respectively. These values represent 1.66-, 1.42-, and 2.51-fold increase compared to non-catalytic process, respec­tively. Besides this good performance, the catalyst maintained the effectiveness after successive recycling being a promising candidate for research applications in oxidation of lignins from other sources.

Ambient temperature bacteria (mesophiles)

I

Elution of Metals usingMetal Specific Chelating Resin

Extremely

thermophilic Archaea

14.1 Conclusion

Bioextraction has been identified as a potential technology for effective extraction and removal of metals in metal overburdened sites, hence relieving the environ­mentally stressed ecosystem. Integration of bioextraction and solid phase extrac­tion methodology helps to recover the heavy metal back by encapsulating precious metals from biomass using metal selective chelating resin, making this approach greener and constructive for mankind. The chapter presents the simplistic under­standing of this environmentally benign alternative approach.

An increased use of biofuels would contribute to sustainable development by reducing greenhouse gas emissions and the use of non-renewable resources. In recent years, it has been suggested that instead of traditional feedstocks, cellulosic biomass (cellulose and hemicellulose), including sugarcane bagasse could be used as an ideally inexpensive and abundantly available source of sugar for fermenta­tion into transportation fuel ethanol. The efficiency of biomass conversion into ethanol depends upon the ability of the microorganism used in the process to utilize these diverse carbon sources and the amount of fraction present in biomass. The cost of ethanol production from sugarcane bagasse is relatively high based on current technologies.

As the price of current ethanol feedstocks (e. g. Corn) is estimated to increase, lignocellulosic materials remain the only viable candidate to serve as renewable feedstock for ethanol production. There are huge amounts of wheat straw that are currently burnt in the field or wasted otherwise which can be used as low value raw material for ethanol production. Despite extensive technological advances in ethanol production from lignocellulose feedstocks over the last few decades, the price of the second-generation ethanol is still high and remains around $2.65/ gallon [101, 102]. This high price is because of some technological impediments encountered in all the different steps of the process. Pretreatment is estimated to account for 33% of the total cost [187]. The current leading pretreatment methods for lignocellulosic materials are capital intensive. Economical comparison showed that there is little differentiation between studied pretreatment methods as for instance; low cost pretreatment reactors are counterbalanced by higher cost of catalyst and/or ethanol recovery [42]. Development of less energy intensive and more effective pretreatment methods allowing lower amount of enzymes loading can substantially decrease the total cost of cellulosic ethanol.

The utilization of lignocellulosic biomass for bioethanol production necessi­tates the production technology to be cost-effective and environmentally sustain­able. Considering the evolution and need of second-generation biofuels, rice straw appears to be a promising and potent candidate for production of bioethanol due to its abundant availability and attractive composition. Biological conversion of rice straw into fermentable sugars, employing hydrolyzing enzymes is, at present the most attractive alternative due to environmental concerns. Although there are several hindrances in the way of developing economically feasible technology due to its complex nature, high lignin, and ash content, work is going on to develop an efficient pretreatment method to remove unwanted portions so as to get readily available sugars and a considerable success has been achieved till date. The available statistics show that the need of bioethanol for the transport sector could be met by using rice straw. Approaches in both process engineering and strain engineering still have to be carried out to circumvent the difficulties of xylose and glucose co-fermentation and to improve the system efficiency. A very balanced and intelligent combination of pretreatment, hydrolysis, and the fermentation process has to be selected for maximum efficacy of the process. With the advent of genetically modified yeast, synthetic hydrolyzing enzymes, other sophisticated technologies and their efficient combination, the process of bioethanol production employing rice straw will prove to be a feasible technology in the very near future.

Dimethyl formamide (DMF), with a high selectivity to delignification, was used as a solvent for pulping. Many lignocellulosic materials, such as bagasse [144, 173], wheat straw [143], rich straw [174] and canola stalks [175], have been subjected to pulping in this context and the main operation parameters (time, temperature concentration, liquid to wood ratio, etc.) were optimized.

DMF pulping has many advantages such as obtained pulp with more hemi — celluloses, less cellulose degradation, high yield, low residual lignin content, high brightness and good strength. The pulp produced was easy to be bleached and the yield after bleaching was higher than the yield of kraft pulp [173]. For example, pulps with high mechanical properties comparable to kraft pulp were produced under such conditions, i. e., at 210°C for 150 min with 50% DMF [143].

The relatively high selectivity of acetone fractionation process is ascribed to the unique chemical mechanism. In most organosolv fractionation processes, protic solvents (such as alcohols with the addition of acids or bases) result in the main delignification and degradation of carbohydrate under certain conditions. However, in an aprotic DMF solvent, the main and only reaction during frac­tionation process is delignification. The reaction results in the cleavage of car­bohydrate-lignin ether linkage and hydrolysis of fi-O-4 and a-O-4 bonds of lignin to form small fragments of lignin. In addition, DMF plays an important role in protecting carbohydrates [173, 176].

It should be noted that other than the organic solvents mentioned above, phenols, esters, ammonia, amines, formamide, dioxane, etc., have also been used to fractionation of a variety of lignocellulosic materials, but these processes are mainly investigated to production of pulps in a laboratory scale presently [2, 4, 13].

The process of liquefying spruce and birch in SCM was achieved in a simple, single-step reaction that occurs in a batch system until the desired conditions are met, over a few minutes as shown in Fig. 13.1.

Under the batch system studied, methanol goes from a fluid density of 0.791 g/l to a density of 0.325 g/l under supercritical conditions, allowing for complete penetration and saturation of the wood particles in the reactor. As the SCM de — polymerizes and solubilizes the major wood components, it reacts with the derived oligomers and monomers, by methanolysis [21, 25]. The mass balance calcula­tions, based on Eq. 13.1, yielded a conversion constant of solid Sitka spruce into bio-oil of 92 wt%, while Alaskan birch yielded 95 wt%.

Residue(%) = W1/W0 x 100 (13.1)

where W0 is the original amount of biomass introduced into the reactor and W1 is the insoluble solids left after supercritical methanol treatment.