D2.1 Lignocellulosic Biomass: Supply and characterization

Transcription

1 Ref. Ares(2015) /09/2015 Project Number: Start date of the project (duration): 1 st May 2015 (36 months) D2.1 Lignocellulosic Biomass: Supply and characterization Organisation name of lead contractor: Técnicas Reunidas Other organisations involved: CENER Authors: Alvaro Pallares, Clemente García Due date of deliverable: M4 Submission date: 01/09/2015 Dissemination Level: PU Copyright 2015 ButaNexT Consortium

2 List of reviewers Issue Date Comments by Implemented by v.final 31/08/2015 v.2 v.1 28/08/2015 CENER v.0 13/08/2015 2

3 Table of Contents EXECUTIVE SUMMARY... 4 INTRODUCTION Chemical characterization of the three feedstock s Feedstocks Wheat Straw Miscanthus Solid urban waste Analytical equipment HPLC and UHPLC SOXHLET EXTRACTOR AUTOCLAVE ANALYTICAL BALANCE TOTAL ORGANIC CARBON ANALYZER ROTARY EVAPORATOR UV-VIS SPECTROPHOTOMETER FREEZER STORAGE ROOM QUARTERING JONES VIBRATORY RING MILL SIEVE EQUIPMENT Preparation of samples for compositional Analysis protocol Samples drying procedure Samples milling procedure Municipal Solid Waste sieving (MSW) Stored samples Carbohydrates characterization protocol Lignin characterization protocol Ash characterization protocol Moisture or total solids content protocol Chemical characterization results Conclusions Biomass Pre-treatment (Preliminary) References

4 EXECUTIVE SUMMARY ButaNexT aims to develop and validate a more economically and environmentally sustainable process for biobutanol production by using several biomass sources (wheat straw, organic fiber from municipal solid waste and miscanthus, ), a two-step pretreatment process and enzyme cocktails bespoke to work synergistically with Clostridium. TR has performed the characterization of the three different types of biomass and also the two-step pretreatment process that releases hemicellulose and cellulose from recalcitrant feedstocks for further enzymatic and/or fermentation processing. The first step is the characterization of the three biomass sources. In this document methods and laboratory equipment used for the biomass characterization have been collected and described. After the characterization of the three biomass sources, miscanthus showed the highest content in glucose (44,8%) and in insoluble lignin (16,3%), whereas wheat straw showed the highest content in xylose (27,8%). Finally it is important to remark that due to the high content in inert material (plastic, stones, others) of the solid urban waste, a sieving separation of inert material was required to perform the analysis, furthermore the sample showed the highest content in ash (51,9%) and the lowest content in glucose (9,4%) and xylose (2,5%). 4

5 INTRODUCTION The ButaNext project aims to develop a cost efficient and environmentally friendly process for biobutanol production from sustainable feedstocks. The objective of this deliverable is the characterization of the three feedstock s supplied by CENER, representative samples of each of the three pre-selected lignocellulosic biomass feedstocks have been analyzed by Tecnicas Reunidas, in order to accurately determine their physicochemical characteristics. The techniques used during this task have been based on the characterization standards of U.S. National Renewable Energy Laboratory (NREL) for different bio-samples of lignocellulose biomasses. These procedures have been adapted from NREL standard TP (1), TP (2) and TP (3), to determine the concentration of sugars (xylose and glucose), acid insoluble lignin, acid soluble lignin, extractives and moisture. An overview of the laboratory equipment used for characterization is also included. 5

6 1. Chemical characterization of the three feedstock s Biomass, protocols and equipment used by TR in the chemical characterization are described below Feedstocks CENER was responsible for managing and coordinating the supply of the three selected lignocellulose biomass candidates to TR. CENER supplied last June and July 20 Kg in big-bags of wheat straw from Orvalaiz Sociedad Cooperativa Agraria (Navarra, Spain), 20 Kg in big-bags of miscanthus from Sica Bourgogne Pellets (Aiserey, France) and 10 Kg of the Organic Fibre (treated organic fraction from municipal solid waste) Tratamientos Ecológicos del Noroeste (TEN SL) Wheat Straw This by-product of agriculture consists of wheat fiber residues about 10 to 20 cm long and 0,5 to 1 cm thick Miscanthus Figure 1 Wheat straw This by-product of agriculture consists of miscanthus fiber residues about 5 to 10 cm long and 1 to 2 cm thick. Figure 2 Miscanthus 6

7 Solid urban waste Municipal waste fraction "Rest" (unsorted waste) come from a sanitation process called thermal waste treatment (TWT). Residues after grinding for homogenization are introduced into a rotary autoclave to be subjected to a steam bath at high temperature in a high pressure environment, then the fractions are separated in ferrous metals, non-ferrous metals, plastics and organic fiber which is the feedstock used in the project. In this picture pieces of glass, stones and plastics can still be observed after TWT in a representative sample supplied. Figure 3 Solid Urban Waste 7

8 1.2. Analytical equipment TR comprehensive analysis laboratory has been able to improve, through continuous and precise adjustments, the performance of several analysis techniques based on the characterization standards of National Renewable Energy Laboratory U.S (NREL) for different bio-samples of lignocellulose biomasses (1) (2) (3) Biomass composition is typically expressed in terms of structural carbohydrates (cellulosic and hemicellulosic content), lignin (soluble and insoluble), ash and moisture content. Structural carbohydrates and lignin make up the bulk of most feedstocks and often represent the most interesting portions HPLC and UHPLC TR s laboratory is equipped with a high-performance liquid chromatograph with high efficiency detectors and Refractive Index Perkin Elmer Model flex LC (HPLC), and also an ultra-high efficiency liquid chromatograph with refractive Index detectors, LC flex Perkin Elmer Model (UHPLC). The two equipments are used to quantify sugars, furfural derivatives and organic acids from biomass samples, using the refractive index detector to determine not chromophore-containing compounds (sugars) and diode array for chromophore-containing compounds (furfural) SOXHLET EXTRACTOR Figure 4 HPLC and UHPLC The soxhlet extractor is a piece of glass used in laboratory for compounds extraction (generally known as extractives) from a solid sample, generally with lipid nature, using a related solvent (i.e.: water, ethanol,.). The equipment is operated cyclically, to extract the necessary concentrations of a given compound. When the solvent is evaporated, the solvent rises to the upper area where it is condensed; and falls back to the camera of solvents. It will separate the compounds until it reaches a desired concentration. 8

9 Figure 5 Soxhlet extractor AUTOCLAVE An autoclave is a metal pressure container with thick walls and a seal that allows working at high pressure for an industrial reaction, cooking or and steam sterilization. The construction should be such as to resist the pressure and temperature built inside. In this project it is used for sample hydrolysis before the extraction process. Figure 6 Autoclave Selecta Micro ANALYTICAL BALANCE An analytical balance is designed for measuring small masses, initially in the lower milligram range. The characteristics of the balance of TR are: Weighing capacity: 220 g Readability: 0.1 mg Tare range (subtraction) 100% of the maximum weighing capacity Weighing range: 0.01 g to 220 g Linearity deviation: 0.2 g 9

10 Figure 7 Sartorius analytical balance cp224s TOTAL ORGANIC CARBON ANALYZER TR is equipped with a total organic carbon (TOC) analyser. It is equipment used for the analysis of carbon content of liquid samples. The TOC binds to hydrogen, oxygen, nitrogen or other elements to form organic compounds. The inorganic carbon (IC or ICT) is the structural basis of inorganic compounds such as carbonate and bicarbonate. The sum of the two forms of carbon (organic and inorganic) is the total carbon (TC) and the relationship between them is expressed as TOC = TC-IC. The TOC-V CSH measured amounts of total carbon (TC), inorganic carbon (IC) and total organic carbon (TOC). The method used for the analysis OCD is the oxidative combustion. This equipment also has the ability to measure total nitrogen (TN) that is equipped with a TNM-1 module oxidative combustionchemiluminescent detection. In our case it is used for determining the total nitrogen present in the biomass samples. Figure 8 Total organic carbon analyzer SHIMADZU TOC-V CSHA ASI-V AUTOSAMPLER and nitrogen column tnm-1 10

11 ROTARY EVAPORATOR A rotary distillation apparatus associated with a water bath is used mainly for separating the solvent accompanying the solute of interest by gentle evaporation at reduced pressure, or, for fractional distillation. It is used in this project to evaporate samples obtained from the hydrolysis of biomass. Figure 9 ROTARY EVAPORATOR BUCHI UV-VIS SPECTROPHOTOMETER TR is equipped with a spectrophotometer Perkin Elmer Model 35 LAMDA S10 autosampler, the instrument has the ability to project a beam of monochromatic light through a sample and measuring the amount of light that is absorbed by the sample. This allows the operator to perform two functions: to provide information about the nature of the substance in the sample and indirectly indicate the amount of the substance that interests us is present in the sample. This equipment is used to quantify the amount of soluble lignin present in the biomass samples. Figure 10 UV-VIS Spectrophotometer Perkin Elmer Model 35 LAMDA S10 autosampler 11

12 FREEZER TR is equipped with a 450 l freezer that is used to preserve biomass samples, liquor and solids from hydrolysis at -20 C prior to characterization or shipping. Figure 11 HQ Freezer STORAGE ROOM TR is equipped with a storage room to preserve in optimal conditions of humidity and temperature biomass samples. Figure 12 Storage room QUARTERING JONES TR is equipped with a JONES for quartering and homogenizing samples. The JONES makes the quartering a systematic process; the whole sample is passed through the Jones for successive passes, after a few passes a representative sample is collected to use in other stages. Figure 13 JONES 12

13 VIBRATORY RING MILL TR is equipped with a hammer mill, Disc mill, ball mill and Vibratory ring Mill. The vibratory ring mill produce obtain small particle sizes "analytical grade" for medium-hard materials, hard, brittle and fiber. It could be used for coal, coke, corundum, slag, ores, metal oxides, etc. The results are reproducible in equal times. The material is reduced by effect of pressure, shock and friction of the rings within the vessel using a circular motion in the horizontal plane as a result of centrifugal force. The grinding set (vessel-lid-rings) once charged is fixed on the vibrating element by a snap-hook, the top of the protective cage is lowered and is started by switch for a preset time, and it is dry grinding. Figure 14 Vibratory mill ring and cage protection SIEVE EQUIPMENT TR is equipped with ASTM standard sieve equipment. The sieving method involves placing a series of sieves on a blind plate, a sieve over another one increasing mesh size. An amount of a homogenous dry weight sample is transferred from the top sieve to the bottom. The whole system is shaken by a rotary and vibration motion. Then the weight of retained material on each sieve is determined. Results of particle size passing through each mesh are expressed in percent weight. Figure 15 ASTM Sieves 13

14 1.3. Preparation of samples for compositional Analysis protocol This procedure describes a preparation of samples for compositional analysis based on NREL/TP (2) protocol in order to convert a variety of biomass samples into a uniform material suitable for compositional analysis. This section describes the methods for drying, size reduction, obtaining samples with a uniform particle size and representative sampling of biomass materials. Biomass homogeneization Biomass Drying Biomass Milling Biomass Characterization Municipal Solid Waste Homogeneization Municipal Solid Waste Drying Municipal Solid Waste Sieving Municipal Solid Waste Milling Municipal Solid Waste Characterization Figure 16 Sequence biomass sample preparation for Characterization Samples drying procedure This procedure has been adapted from NREL standard TP (2). After selecting the container bullet for the feedstock sample drying, the sample container was dried at 45 C for 3 h in an oven, then it was placed and cooled to room temperature in a desiccator and weighted, the weight was recorded. A feedstock sample was placed into the dried container, the container was weighted and the weight was recorded, then it was placed in the drying oven maintaining the temperature at 45 C for 48 h. After 48 h the container was removed from the drying oven and placed and cooled in a desiccator to room temperature. After cooled it was weighted and the weight obtain was recorded. The sample was returned to the drying oven, maintaining the temperature at 45 C for 4 h. The container was removed and placed in the desiccator and the weight was recorded, then the container was returned to the drying oven at 45ºC for 1h, the sample was removed from the drying oven and placed in the desiccator to cooled it to room temperature, the sample was weighted, finally the weight did not change more than 1%. With the last weight obtained the total solids was calculated Samples milling procedure 20 gr of dried wheat straw and miscanthus were fed and milled using the vibratory ring mill for 1 minute. The entire sample obtained overpassed a sieving with 2 mm mesh. The prepared samples were stored in sealable polyethylene bags and kept at -20 C until the characterization assays. This procedure has been adapted from NREL standard TP (2). 14

15 Municipal Solid Waste sieving (MSW) In order to perform a chemical characterization of the sample of MSW a visual inspection was made, Large size of inert materials (glass, stones and plastics) were observed, it was decided to process and analyze only the solids fraction below 4 mm (maximum size supported by vibratory ring mill). Method: The Urban Solid Waste sample was homogenized with a quartering JONES (Figure 13) and dried by NREL method, the different fractions obtained after sieving were weighed and the fraction less than 4 mm was milled with the vibratory ring mill for further analysis. The prepared sample was stored in sealable polyethylene bag and kept at -20 C until the characterization. In figure 15 different fraction obtained after sieving are showed. Solid urban waste sieving (A) ASTM 3-mesh 6,3 mm (B) ASTM 5-mesh 4 mm (C) ASTM 7-mesh 2,8 mm (D) ASTM 10-mesh 2 mm (E) ASTM 14-mesh 1,4 mm (F) ASTM 18-mesh 1 mm 15

16 (G) BLIND Figure 17 Municipal Solid waste sieving sequence In figure 17 the weight rejection is showed, it is important to remark the high content in stones, plastics and glass present in the sample are showed in picture A and picture B (figure 17), these fractions represents the 52% of the total sample weight and cannot be processed for analysis, a small part of <4mm fraction picture A and B was organic matter, a manual separation of the original sample (described below) was performed to quantify what percentage of the municipal solid waste were inert materials. ASTM nº mesh size (mm) weight rejection (g) % Rejection BLIND 0,000 88,3 17,73% 18 1,000 25,7 5,16% 14 1,400 39,9 8,01% 10 2,000 42,5 8,54% 7 2,800 43,0 8,64% 5 4,000 54,6 10,97% 3 6, ,9 40,95% Figure 18 MSW particle size distribution Manual separation of stones, plastic and glass was performed in order to know the weight of these fractions of the municipal solid waste, fractions were weighted after performing the separation, the results and the appearance of materials are shown in Figure 17 and Figure 19. It notes that the percentage of inert materials in the sample is 40% and only the weight of glass is 25% of the total, so it is essential for characterization and pre-treatment of the sample perform a preliminary step to remove such materials. For the characterization the <4 mm fraction after sieving was removal, through this method 10% of organic matter is lost. 16

17 Figure 19 Inert materials in municipal solid waste Stored samples Municipal Solid Waste Weight (g.) ww Percentage Glass 83,6 26% Stones 30 9% Plastic 15 5% organic fraction 196,4 60% % Figure 20 Municipal solid waste weight distribution The three supplied feedstocks were stored in a storage room (Figure 12) with controlled humidity and temperature. Milled samples were stored in sealable polyethylene bags and kept at -20 C in a freezer (Figure 11) until the characterization Carbohydrates characterization protocol The carbohydrates making up a major portion of biomass samples are polysaccharides composed primarily of glucose, xylose, arabinose, galactose and mannose subunits. These polysaccharides, mainly cellulose and hemicellulose, can be hydrolyzed to their component sugar monomers by sulphuric acid in a two-stage hydrolysis process. This procedure has been adapted from NREL standard TP (1). For a correct biomass characterization, it is imperative to perform a pre-extraction with ethanol or water in a Soxhlet for hours, after this extraction a hydrolysis step is carried out. During hydrolysis the polymeric carbohydrates are hydrolyzed into the monomeric forms, which are soluble in the hydrolysis liquid, and they could be measured by High Performance Liquid Chromatography (HPLC) using a Biorad Aminex HPX-87P column equipped with the appropriate guard column. To this purpose it is necessary: 1. To prepare a set of sugar recovery standards (SRS) that will be taken through the remaining hydrolysis and used to correct for losses due to sugar degradation during dilute acid hydrolysis 17

18 (this will give an estimate of the amount of each individual sugar degraded during the hydrolysis procedure). 2. To prepare a series of calibration standards (between 10 and 1000 mg/ml of each sugar) containing the compounds that are to be quantified, four calibration points are calculated. 3. To create a calibration curve by linear regression analysis for each sugar to be quantified and determine the concentration in mg/ml of the sugars present in each solution analyzed by HPLC. 4. To calculate the concentration of the polymeric sugars from the concentration of the corresponding monomeric sugars, using an anhydrous correction of 0.88 (weight ratio between the monomer and the polymer) for C-5 sugars (xylose and arabinose) and a correction of 0.90 for C-6 sugars (glucose, galactose and mannose) (weight ratio between the monomer and the polymer). 18

19 1.5. Lignin characterization protocol This procedure has been adapted from NREL standardtp (1). The lignin fractionates into acid insoluble material (AIL) and acid soluble material (ASL). The total lignin in a biomass sample includes both acid-soluble lignin and lignin in the acid insoluble residue. The Acid Soluble Lignin (ASL) is low molecular weight lignin that solubilized in the acidic hydrolysis solution which is measured by using a UV-Visible spectrophotometer: 1. Take an aliquot of the liquor obtained during the hydrolysis step and measure the absorbance of this sample at an appropriate wavelength on the UV-Visible spectrophotometer running a background of 4% sulphuric acid. 2. The dilution of the samples is necessary to bring the absorbance into the range of , recording the dilution. 3. Each sample is analyzed in duplicate, at minimum. 4. By the application of Lambert-Beer Law, the amount of acid soluble lignin, in mg/ml, is measured. Benzoic acid is used as a System Suitability Standard of the assay. The acid insoluble material, considered high molecular weight lignin, may also include ash and proteins, which must be accounted for during gravimetric analysis. The analysis of the sample for Acid Insoluble Lignin (AIL): 1. Use deionized water to quantitatively transfer all remaining solids out of the pressure tube (after the hydrolysis stage) into a filtering crucible and to wash the solids. 2. Dry the crucible and acid insoluble residue at 105ºC, and then place the residue in the muffle furnace at 575ºC for 24 hours or to constant weight. The weight of the residue corresponds to the lignin insoluble fraction of the sample Ash characterization protocol The amount of acid insoluble ash is not equal to the total amount of ash in the biomass sample. The amount of inorganic material in biomass, either structural or extractable, must be measured as part of the total composition. The ash content is a measure of the mineral content and other inorganic matter in biomass and it may interfere with acid hydrolysis. The tests method performed in our laboratory (adapted from NREL standard TP ) (4) covers the determination of ash, expressed as the percentage of residue remaining after dry oxidation at high temperature (550 to 600ºC) of the initial sample of biomass Moisture or total solids content protocol Biomass samples can contain large and varying amounts of moisture, which can change quickly when exposed to air. The moisture content is a measure of the amount of water (and other components volatilized at 105ºC) present in a sample. It is an important data (provided by the R&D Department and adapted from NREL/TP ) (3)because the results of the chemical analyses of biomass samples are typically reported on a 105ºC dry weigh basis. 19

20 2. Chemical characterization results In order to properly characterize the three selected feedstocks, two representative samples of each one were collected, and studied separately. The results, shown in figure 20, were obtained from averaging the two characterization results, together with the standard deviation. Miscanthus has the highest content in glucose (44,8 %) and in insoluble lignin (16,3 %), Wheat straw has the highest content in xylose (27,8%). It is important to remark the low concentration of glucose (9,4%) and xylose (2,5%) of the municipal solid waste, furthermore it contains a very high percentage of ash (51,9%) and total lignin (18%) due to the high inert materials content as it is seen in figure 15. Feed Stock Wheat Straw Miscanthus Municipal Solid Waste Glucose %ww Chemical Characterization Xylose %ww Lignin %ww AIL ASL Total Ash %ww Moisture %ww Average 44,8 27,8 9,8 3,0 12,8 4,7 8,0 SD 1 0,0 0,1 0,0 0,0 0,1 0,0 0,3 Average 51,7% 23,2% 16,3% 1,8% 17,9% 1,6% 9,0% SD 0,6% 0,3% 0,3% 0,1% 0,1% 0,0% 0,0% Average 9,4% 2,5% 15,0% 3,2% 18,0% 51,9% 5,8% SD 0,0% 0,0% 0,4% 0,0% 0,0% 0,1% 1,6% Figure 21 Biomass chemical characterization 20

21 3. Conclusions Through the characterization of the three selected samples (wheat straw, miscanthus and organic fraction from municipal solid waste) we have observed that the biomass with highest glucose is Miscanthus with 51.7%, closely followed by wheat straw with only 44.8%.. Wheat straw has the highest content of xylose( 27,7 %). On the other hand, the high content and size of inert materials (plastic, stone, glass...) present in the sample of Municipal solid waste was surprising, furthermore this feedstock source has the lowest content in glucose (9,2 %) and xylose (2,5 %). It also is the biomass with highest ash content due to the high amount of intert materials. On the other hand the following problems were found in the characterization of samples: - Due to the hardness and length of miscanthus manual size reduction was necessary before processing it with the vibratory ring mill. - Moreover due to the high content of inert materials in municipal solid waste it was decided to study the weight and size of the inert materials. A manual separation of stones, plastics and glasses was performed and the different fractions obtained were weighted. The results showed that 40% of the sample corresponded to inert materials. Additionally a sieving study was performed. The largest materials observed (>4 mm) after these study corresponded to inert materials and the total weight rejection after sieving these fraction was 52% (w/w). The conclusion was to use only the fraction lower than 4 mm sieve mesh, because this fraction has the lowest content of glass, plastic and stones. Despite the problems found especially with municipal solid waste handling and conditioning all characterizations were performed successfully. 4. Biomass Pre-treatment (Preliminary) In order to produce enough pre-treatment Biomass substrates for the Baseline strain selection task 2.4, TR designed a preliminary two-step pre-treatment method. It is described below. Physical pre-treatment: myscanthus and wheat straw were micronized with a vibratory ring mill, municipal solid waste was sieved in order to reject inert materials (>4mm mesh) before milling. Chemical pre-treatment: milled myscanthus and wheat substrates were hydrolysed using a weak acid solution in a reactor at 120ºC for 90 minutes, municipal solid waste was hydrolysed using a weak acid solution in a reactor at 60ºC for 5 hours. This pre-treatment is preliminary and is not optimized yet. It will be used for the first steps of the project. The optimized pre-treatment will be reported in the following project deliverables: D.2.2 Pre-treatment micronizing prototype deliverables and D2.3 Thermochemical pre-treatment. 21

22 5. References 1. A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, D. Templeton. Determination of Structural carbohydrates and Lignin in Biomass THECNICAL REPORT NREL/TP NATIONAL RENEWABLE ENERGY LABORATORY Laboratory Analytical procedure. 2. B. Hames, R. Ruiz, C. Scarlata, A. Sluiter, J. Sluiter, D. Templeton. Preparation of Samples for Compositinal Analysis NREL/TP NATIONAL RENEWABLE ENERGY LABORATORY LABORATORY ANALYTICAL PROCEDURE. 3. A. Sluiter, B. Hames, D. Hyman, C. Payne, R. Ruiz, C. Scarlata, D. Templeton. DETERMINATION OF TOTAL SOLIDS IN BIOMASS AND TOTAL DISSOLVEVED SOLIDS IN LIQUID PROCESS SAMPLES NREL/TP NATIONAL RENEWABLE ENERGY LABORATORY LABORATORY ANALYTICAL PROCEDURE. 4. A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, and D. Templeton. Determination of Ash in Biomass NREL/TP National Renewable Energy Laboratory Laboratory Analytical Procedure (LAP). 22