• Feedstocks Analysed at Celignis
    Forest Residues

Background on Forest Residues

Wood with a smaller diameter than 70 mm (branches and the tops of trees) is generally not harvested in roundwood operations and is left on the forest floor.

Forest residues represent a highly heterogeneous mix of biomass. In addition to the tree-tops and branches there will also be a significant amount of foliage.

The constitution and total quantity of forest residues on any particular site is dependent on: the age of the tree; the species; the tree-spacings; and the wood assortment harvested. Depending on these, the amount of forest residues can vary from 50 to 100 oven dry tonnes per hectare.

The biomass obtained after thinning operations is also of relevance. Due to their small size, these trees, particularly first thinnings, are often not used in sawmills or for pulpwood.

Analysis of Forest Residues at Celignis



Celignis Analytical can determine the following properties of Forest Residues samples:



Lignocellulosic Properties of Forest Residues

Cellulose Content of Forest Residues

The composition of forestry residues will depend on: the tree species; the time of year; and the method used for collecting the residues, among other factors.

Given that foliage and bark typically contain lower cellulose contents than wood, a greater mass proportion of stem wood will favour an increased cellulose content in the harvested residues.

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Hemicellulose Content of Forest Residues

As with cellulose, the hemicellulose content of forestry residues will depend on the mass proportions that the different tree fractions (leaves, bark, tops, branches, stem wood) contribute to the total mass of harvested residues.

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Lignin Content of Forest Residues

Increased proportions of bark and stem wood in the harvested residues will favour an increased lignin content.

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Starch Content of Forest Residues

Starch can be an important constituent of forest residues as it tends to be in higher concentrations in the foliage and bark fractions of trees.

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Uronic Acid Content of Forest Residues

The uronic acid content and composition of forestry residues will depend on the mass proportions that the different tree fractions contribute to the total mass of harvested residues. For instance, bark tends to have the highest uronic acid content, followed by leaves, with lower amounts in the stem wood.

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Enzymatic Hydrolysis of Forest Residues

We can undertake tests involving the enzymatic hydrolysis of Forest Residues. In these experiments we can either use a commercial enzyme mix or you can supply your own enzymes. We also offer analysis packages that compare the enzymatic hydrolysis of a pre-treated sample with that of the native original material.

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Bioenergy Properties of Forest Residues

Ash Content of Forest Residues

While most wood has a relatively low ash content, foliage can contain a significant amount of ash. Hence, a greater mass proportion of foliage in the harvested residues will lead to an increased ash content in the sample.

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Heating (Calorific) Value of Forest Residues

The heating value of forestry residues will depend on the mass proportions that the different tree fractions (leaves, bark, tops, branches, stem wood) contribute to the total mass of harvested residues.

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Ash Melting Behaviour of Forest Residues

Ash melting, also known as ash fusion and ash softening, can lead to slagging, fouling and corrosion in boilers which may reduce conversion efficiency. We can determine the ash melting behaviour of Forest Residues using our Carbolite CAF G5 BIO ash melting furnace. It can record the following temperatures:

Ash Shrinkage Starting Temperature (SST) - This occurs when the area of the test piece of Forest Residues ash falls below 95% of the original test piece area.

Ash Deformation Temperature (DT) - The temperature at which the first signs of rounding of the edges of the test piece occurs due to melting.

Ash Hemisphere Temperature (HT) - When the test piece of Forest Residues ash forms a hemisphere (i.e. the height becomes equal to half the base diameter).

Ash Flow Temperature (FT) - The temperature at which the Forest Residues ash is spread out over the supporting tile in a layer, the height of which is half of the test piece at the hemisphere temperature.



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Major and Minor Elements in Forest Residues

Examples of major elements that may be present in Forest Residues include potassium and sodium which are present in biomass ash in the forms of oxides. These can lead to fouling, ash deposition in the convective section of the boiler. Alkali chlorides can also lead to slagging, the fusion and sintering of ash particles which can lead to deposits on boiler tubes and walls.

We can also determine the levels of 13 different minor elements (such as arsenic, copper, and zinc) that may be present in Forest Residues.

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Analysis of Forest Residues for Anaerobic Digestion



Biomethane potential (BMP) of Forest Residues

At Celignis we can provide you with crucial data on feedstock suitability for AD as well as on the composition of process residues. For example, we can determine the biomethane potential (BMP) of Forest Residues. The BMP can be considered to be the experimental theoretical maximum amount of methane produced from a feedstock. We moniotor the volume of biogas produced allowing for a cumulative plot over time, accessed via the Celignis Database. Our BMP packages also involve routine analysis of biogas composition (biomethane, carbon dioxide, hydrogen sulphide, ammonia, oxygen). We also provide detailed analysis of the digestate, the residue that remains after a sample has been digested. Our expertise in lignocellulosic analysis can allow for detailed insight regarding the fate of the different biogenic polymers during digestion.



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Physical Properties of Forest Residues



Bulk Density of Forest Residues

At Celignis we can determine the bulk density of biomass samples, including Forest Residues, according to ISO standard 17828 (2015). This method requires the biomass to be in an appropriate form (chips or powder) for density determination.



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Particle Size of Forest Residues

Our lab is equipped with a Retsch AS 400 sieve shaker. It can accommodate sieves of up to 40 cm diameter, corresponding to a surface area of 1256 square centimetres. This allows us to determine the particle size distribution of a range of samples, including Forest Residues, by following European Standard methods EN 15149- 1:2010 and EN 15149-2:2010.



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Publications on Forest Residues By The Celignis Team

V. P. Zambare, S. S. Nilegaonkar and P. P. Kanekar (2014) Scale up production of protease using Pseudomonas aeruginosa MCM B-327 and its detergent compatibility, Journal of Biochemical Technology 5(2): 698-707

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The Maximum Protease Activity Was Obtained From P. Aeruginosa MCM B-327 With Soybean Meal 1%, Tryptone 1%, Initial Medium PH 7, Agitation Rate 250 Rpm, Aeration Rate 0.75 Vvm And Fermentation Temperature 30 C, Under Submerged Fermentation Conditions (SmF). The Protease Productivity At 10 And 120L Fermenters Was Found To Be 16,021 And 9,975 UL-1h-1 Respectively. Kinetics Of Cell Growth Revealed That Specific Cell Growth Rate Was 0.025 H-1. Protease Was Active And Stable At Different PH, Temperatures, In Anionic, Cationic And Non-Ionic Detergent Additives, As Well As In Commercial Detergents. The Protease Exhibited Blood Stains Removing Performance Indicating Its Potential In Detergent Industry. The Dried Ammonium Sulphate Precipitated Protease Was Stable At Room Temperature For A Period Of One Year. The Protease Has Shown Properties Suitable For Its Application In Detergents. The Results Contribute To Basic Knowledge And Application Of Protease From P.Aeruginosa To Detergent Industry. The Studies Will Help To Optimize The Production Of This Protease For Biotechnological Applications.

V.P. Zambare, Lew P. Christopher (2012) Optimization of enzymatic hydrolysis of corn stover for improved ethanol production, Energy Exploration & Exploitation 30(2): 193-205

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Response surface methodology (RSM) was used to optimize the enzymatic hydrolysis of corn stover (CS), an abundant agricultural residue in the USA. A five-level, three-variable central composite design (CCD) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.16.0), solids loadings (6.623.4%), and enzyme loadings (6.6?23.4 FPU g?1 DM) on glucose yield from thermo-mechanically extruded CS. The extruded CS was first hydrolyzed with the crude cellulase of Penicillium pinophilum ATCC 200401 and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance (ANOVA) showed a coefficient of determination (R2 ) of 0.82. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU g?1 DM, the enzymatic hydrolysis of pretreated CS produced a glucose yield of 57.6% of the glucose maximum yield which was an increase of 10.4% over the non-optimized controls at zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges.

Vasudeo Zambare, Archana Zambare, Debmalya Barh, Lew Christopher (2012) Optimization of enzymatic hydrolysis of prairie cordgrass for improved ethanol production, Journal of Renewable and Sustainable Energy 4(3): 1-8

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Prairie cordgrass (PCG), Spartina pectinata, is considered an energy crop with potential for bioethanol production in North America. The focus of this study was to optimize enzymatic hydrolysis of PCG at higher solids loadings using a thermostable cellulase of a mutant Penicillium pinophilum ATCC 200401. A three variable, five-level central composite design of response surface methodology (RSM) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.16.0), solids loadings (6.6%23.4%), and enzyme loadings (6.623.4 FPU/g dry matter, DM) on glucose yield from a thermo-mechanically extruded PCG. The extruded PCG was first hydrolyzed with the crude P. pinophilum cellulase and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance showed a coefficient of determination (R2) of 0.89. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU/g DM, the enzymatic hydrolysis of pretreated PCG produced a glucose yield of 76.1% from the maximum yield which represents an increase of 15% over the non-optimized controls at the zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges. Furthermore, statistical optimization of solids and enzyme loadings of enzymatic hydrolysis of biomass may have important implications for reduced capital and operating costs of ethanol production.

V. P. Zambare, S. S. Nilegaonkar and P. P. Kanekar (2012) Optimization of Nutritional Factors for Extracellular Amylase Production from Bacillus cereus MCM B-326 Using Response Surface Methodology, Research Journal of BioTechnology 7(4): 58-65

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The optimization of nutritional factors and their concentrations for the amylase production by Bacillus cereus MCM B-326 in submerged fermentation was carried out using response surface methodology (RSM) based on the central composite design (CCD). The design contains a total of 20 experimental trials containing starch, soybean meal and CaCO3 as model factors for three levels. The mutual interaction between these variables resulted into 1.36 fold increase in amylase activity as compared to the mean predicted response at zero level of all variables. Amylase from B. cereus has approximate molecular weight of 40 kDa with optimum activity at pH 7.0 and temperature 30C.

Vasudeo Zambare, Archana Zambare, Kasiviswanathan Muthukumarappan, Lew Christopher (2011) Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing, International Journal of Energy and Environment 2(1): 99-112

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A thermophilic microbial consortium (TMC) producing hydrolytic (cellulolytic and xylanolytic) enzymes was isolated from yard waste compost following enrichment with carboxymethyl cellulose and birchwood xylan. When grown on 5% lignocellulosic substrates (corn stover and prairie cord grass) at 600 C, the thermophilic consortium produced more xylanase (up to 489 U/l on corn stover) than cellulase activity (up to 367 U/l on prairie cord grass). Except for the carboxymethyl cellulose-enriched consortium, thermo-mechanical extrusion pretreatment of these substrates had a positive effect on both activities with up to 13% and 21% increase in the xylanase and cellulase production, respectively. The optimum temperatures of the crude cellulase and xylanase were 600 C and 700 C with half-lives of 15 h and 18 h, respectively, suggesting higher thermostability for the TMC xylanase. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the crude enzyme exhibited protein bands of 25-77 kDa with multiple enzyme activities containing 3 cellulases and 3 xylanases. The substrate specificity declined in the following descending order: avicel>birchwood xylan>microcrystalline cellulose>filter paper>pine wood saw dust>carboxymethyl cellulose. The crude enzyme was 77% more active on insoluble than soluble cellulose. The Km and Vmax values were 36.49 mg/ml and 2.98 U/mg protein on avicel (cellulase), and 22.25 mg/ml and 2.09 U/mg protein, on birchwood xylan (xylanase). A total of 50 TMC isolates were screened for cellulase and xylanase secretion on agar plates. All single isolates showed significantly lower enzyme activities when compared to the thermophilic consortia. This is indicative of the strong synergistic interactions that exist within the thermophilic microbial consortium and enhance its hydrolytic capabilities. It was further demonstrated that the thermostable enzyme-generated lignocellulosic hydrolyzates can be fermented to bioethanol by a recombinant strain of Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.

Vasudeo Zambare (2011) Optimization of amylase production from Bacillus sp. using statistics based experimental design, Emirates Journal of Food and Agriculture 23(1): 37-47

Production of amylase under submerged fermentation Bacillus sp. was investigated using wheat bran, soybean meal and CaCO3 (WSC) medium. Response surface methodology (RSM) was used to evaluate the effect of the main variables, i.e., pH (11.35), temperature (35.16C) and inoculum size (2.95%) on amylase production by applying a full factorial central composite design (CCD). The mutual interaction between these variables resulted into 4.64 fold increase in amylase activity as compared to the non-optimized environmental factors in the basal medium.

V.P. Zambare, Lew P. Christopher (2011) Statistical analysis of cellulase production in Bacillus amyloliquefaciens UNPDV-22, Extreme Life, Biospeology & Astrobiology 3(1): 38-45

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The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology (RSM). Central composite design (CCD) was used to study the interactive effect of fermentation medium components (wheat bran, soybean meal, and malt dextrin) on cellulase activity. Results suggested that wheat bran, soybean meal, and malt dextrin all have significant impact on cellulase production. The use of RSM resulted in a 70% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 11.23 U/mL was obtained in a fermentation medium containing wheat bran (1.03%, w/v), soybean meal (2.43%, w/v), and malt dextrin (2.95%, w/v).

V.P. Zambare, Lew P. Christopher (2011) Optimization of culture conditions for cellulase production from thermophilic Bacillus strain, Journal of Chemistry and Chemical Engineering 5(7): 521-527

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The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology (RSM). Central composite design (CCD) was used to study the interactive effect of culture conditions (temperature, pH, and inoculum) on cellulase activity. Results suggested that temperature and pH all have significant impact on cellulase production. The use of RSM resulted in a 96% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 13 U/mL was obtained at a temperature of 42.24 C, pH of 5.25, and inoculum size of 4.95% (v/v) in a fermentation medium containing wheat bran, soybean meal and malt dextrin as major nutritional factors.

V. P. Zambare (2010) Solid state fermentation production of cellulase from Bacillus sp, International Journal of BioScience, Agriculture and Technology 2(1): 1-6

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Bacillus sp. was cultured in solid-state fermentation (SSF) of wheat straw to produce cellulase. The fermented biomass was harvested after 36 h of SSF at pH 8 and temperature 400C. It was filtered and centrifuged at 10,000 rpm at 4 0C and supernatant was collected as crude enzyme extract. Maximum activity of cellulase (3.7750.13U/ml) was obtained after fermentation of wheat straw (10g) medium containing 0.2g soybean meal, 0.04g corn steep liquor (CSL), 80% moisture content (mineral salt medium, pH 8), 2-mL inoculum, and temperature 40 0C. SSF was found to be more productive than submerged fermentation (SmF) in terms of cellulase yields. The partial purification of cellulase was carried out through (NH4)2SO4 precipitation. The partially purified enzyme produced under SSF had molecular weight of 35 and 45kDa. It was active in a broad pH (4-10) and temperature range (25-550C). The optimum, pH and temperature of Bacillus cellulase were pH 5 and 450C, respectively. At 500C and 600C, the half lives of the partially purified cellulase were 194 and 163 min, respectively. All the results indicated that the Bacillus sp. had a promising application of treatment of agro-wastes and cellulase from Bacillus sp. could be potentially used in biofuel industries.

Vasudeo Zambare (2010) Solid state fermentation of Aspergillus oryzae for glucoamylase production on agro residues, International Journal of Life Sciences 4: 16-25

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Glucoamylase is a well recognized amylolytic enzyme used in food industry, which is generally produced by Aspergillus genus under solid-state fermentation (SSF). This study presents production of glucoamylase by Aspergillus oryzae on the solid surface of rice husk, wheat bran, rice bran, cotton seed powder, corn steep solids, bagasse powder, coconut oil cake, and groundnut oil cake as substrates. Optimization of the SSF media and parameters resulted in a 24% increase in the glucoamylase activity. Optimum glucoamylase production (1986 moles of glucose produced per minute per gram of dry fermented substrate) was observed on wheat bran supplemented with 1%, (w/w) starch, 0.25%, (w/w) urea at pH 6, 100%, (v/w) initial moisture and 300C after incubation 120 hrs. Therefore, A. oryzae can be useful in bioprocessing application for saccharification of agro-residues.

S. S. Nilegaonkar, V. P. Zambare, P. P. Kanekar, P. K. Dhakephalkar, S. S. Sarnaik, N. K. Chandrababu, Rama Rajaram, B. Ramanaiah, T. Ramasami, Y. K. Saikumari and P. Balaram (2006) A novel protease for industrial application, Indian Patent Patent NO. 2471DEL2006

The present invention relates to an extracellular enzyme protease obtained by growing the culture of Pseudomonas aeruginosa MCM B-327 isolated from vermiculture pit soil and deposited in MTCC, IMTECH, Chandigarh with designation MTCC 5270, in production medium of pH 7.0; containing soybean meal and tryptone as raw materials, at 30 C. for 72 h. The organism was also able to produce protease using different agricultural products/byproducts as protein sources. The partially purified non-collagenolytic, calcium independent protease with molecular weight 60 kDa has activity in pH range of 6.0-11.0 and temperature range of 25-65 C.; stability in pH range of 6.0-10.0 and temperature 25-45 C. The protease activity was retained for 8 months when stored at ambient temperature. Ammonium sulphate precipitated enzyme was able to completely dehair animal skins and hides without chemicals like lime, sodium sulphide and calcium.





Examples of Other Feedstocks Analysed at Celignis



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