Cellulosic preprocessing apparatus

About a Cellulosic preprocessing apparatus

About a Cellulosic preprocessing apparatus

Herein we introduce an innovative technique and related equipment for the production of bioethanol from corn stover.
This method (the Kato Saccharide Exposure method; KSE) uses water, heat, and pressure to break down the lignin in corn stover and expose the glucide content, and we developed the KSE equipment based on the principles underlying this method.
 This epoch-making KSE equipment can continuously process corn stover mechanically, without the need for chemicals, to expose the glucide content in as little as 18 seconds.
The energy requirement for the prototype equipment is 33.3kj/kg, and its continuous processing capacity is 23 kg/h.
 To allow processing without the need for chemicals, conventional enzymes (amylase and amylglucosidase) are used to initiate hydrolysis and general-purpose yeast is used to start fermentation, affording one-tank simultaneous fermentation and saccharification without the need for temperature control and with significant savings in terms of cost and time over conventional methods.
Saccharification test results, based on the NREL protocol (NREL/TP-510-42629), demonstrated that processing of corn stover (2mm x 2mm in size) provided 88.9% glucose conversion, which is comparable to the results achieved using genetically modified enzymes.
 This principle is also applicable to the processing of woody biomass; however, saccharification testing revealed a 20.8% saccharification rate, indicating that further optimization of the equipment for handling woody biomass is required.

* Corn stover variety: Pioneer Hybrid Japan 39B29, grown in Shintoku, Hokkaido.

* Composition analysis results for corn stover: glucan 45.0%, xylan 20.9%, galactan 0.0%, arabinan 2.6%, lignin 19.4%, ash 1.9% and extracts 14.3%.

* Preprocessing refers to the series of steps required to prepare cellulosic raw materials for saccharification

Cellulosic preprocessing system

Patent information

International patents

Based on the Patent Cooperation Treaty, we filed an international application (PCT)
and completed the transfer procedure to the designated country.
(The same as the with Japanese Patent Office)
In the US, we have received a patent as a small entity

Technical thesis

(Summary)
Title: A method for the exposure of saccharides in lignocellulose biomass raw materials in 18 seconds

Authors:

Susumu Kato 1) Takaaki Takebe 1) Yukiko Kato 1) Norihiro Terajima 1) Yuka Sugawara 1)

Correspondence to:

Susumu Kato
Kato Biomass Technology Co., Ltd.
Kita 45-jo Higashi 16-chome 1-2, Higashi-ku, Sapporo-shi, Hokkaido 007-0845 Japan
Phone: +81 (11) 783-4565, Fax: +81 (11) 783-4565
E-mail address: s.kato@kato-biomass.com

Abstract:

This is a report on a unique processing method for exposing the saccharide content within lignocellulose biomass raw materials through the disintegration of the tightly interwoven lignin and saccharide components, the underlying principles and methodology, as well as the core element of the apparatus employed.

The production of ethanol from lignocellulose biomass raw material has received much attention due to global efforts to reduce CO2 emissions. However, a number of serious problems remain in relation to saccharification efficiency, processing time, high cost of enzymes and recovery of chemicals. These problems have been discussed with a completely new approach based on an innovative processing method that utilizes a single apparatus for exposing the saccharide content of the raw materials. The process allows the exposing the saccharide content in as little as 18 seconds, and achieves a high saccharification efficiency of 88.9-100% in tests on the disintegrated product.

Main Text:

1.Introduction

2.Theory: Kato Saccharide Exposure method

The research was based on the notion that saccharification of lignocellulose biomass would be more easily accomplished with commonly available enzymes if the saccharide content could be exposed. From this position, a method for the disintegration of the cellulose, hemicellulose, and lignin components in the lignocellulose biomass was designed to expose the saccharide content. The developed method, the Kato Saccharide Exposure (KSE) method, exposes the saccharide content through ….

The apparatus consists of … (Fig. 2) and … (Fig. 3). …This experimental apparatus makes it possible to process lignocellulose biomass raw material so as to produce the exposed saccharides component continuously in as little as 18 seconds from the time the lignocellulose material is fed into the apparatus (maximum productivity, 100 kg/h). Operation of the apparatus requires 33 kJ / kg. It can be increased to allow the processing of raw materials on an industrial scale.

3.Testing

3.1 Test Materials

The analysis used three kinds of herbaceous raw materials with an adjusted moisture content of 28%, corn stover (Fig. 4 Panel A, left), corn dust (Fig. 4 Panel B, left), and broken corn kernels (Fig. 4 Panel C, left). The materials were fed into the device separately and thereafter passed through the three processing zones. Each of these materials was processed to expose the saccharide content through …, thereby producing the KSE-processed corn stover shown in Fig. 4 Panel A (middle), the corn dust in Fig. 4 Panel B (middle), and the broken corn in Fig. 4 Panel C (middle).

The KSE-processed product was then milled to form a fine powder of 105 µm in size using a pin mill (Nara Machinery Co., Ltd., Tokyo, Japan) prior to subsequent saccharification testing. As the purpose of milling is only to facilitate the saccharification testing, the saccharification rate of the KSE-processed powder is not influenced by milling. The pulverized KSE-processed corn stover, corn dust and broken corn kernels are shown in Fig. 4 Panel A (right), Fig. 4 Panel B (right), and Fig. 4 Panel C (right), respectively.

3.2 Raw materials

Details of the raw materials are as follows:

Corn stover (Fig. 4 Panel A, left): Pioneer hybrid Japan 39B29 variety, produced in Shintoku-cho, Hokkaido, Japan (geographical coordinates 43.06472, 142.805109 or 43° 3’ 52.992” North, 142° 48’ 18.3918” East) in 2012. This stover, provided by the Hokkaido Research Organization (a public research institution, Hokkaido, Japan), was pulverized in a cutter mill (Horai Co., Ltd., Osaka, Japan) and classified at 177 µm. The corn stover was kept in the room at approximately 15 °C and at a moisture content of 12 % until further processing.

 Corn dust (Fig. 4 Panel B, left): fragments or fine particles of corn grain or corn cobs generated during transport to Japan of US-grown whole dent corn cobs were kindly provided by Marubeni Corporation (Tokyo, Japan).

Broken corn kernels (Fig. 4 Panel C, left): fragments or fine particles of corn kernels or corn cobs generated during transport to Japan of US-grown dent corn were kindly provided by Marubeni Corporation.

3.3 Component Analysis

The components of these pulverized KSE-processed samples (right side of Fig. 4 Panel A, Panel B, and Panel C) were analyzed by the Hokkaido Research Organization. The analysis was conducted based on the analysis procedures established by the National Renewable Energy Laboratory (NREL) in the USA. The extractives, saccharides, lignin, and ash were analyzed based on the following protocols: 1) extractives (matter extracted by water and alcohol): NREL/TP-510-42619 (11), 2) saccharides and lignin: NREL/TP-510-42618 (12), and 3) ash: NREL/TP-510-42622 (13).

3.4 Saccharification Testing

Saccharification testing of the pulverized KSE-processed samples (right side of Fig. 4 Panel A, Panel B, and Panel C) was also conducted at the Hokkaido Research Organization after the above-mentioned component analysis. The test was based on the NREL/TP-510-42629 protocol (14) established by the NREL.

Each of the samples (right side of Fig. 4 Panel A, Panel B, and Panel C; equivalent to 0.1 g  cellulose) was put into a screw vial container (30 mm in diameter x 65 mm in height, 30 mL total volume), and 5 mL of citric acid buffer, cellulase (Novozymes 50013, 25FPU/g-cellulose, Novozymes A/S, Bagsvaerd, Denmark), beta glucosidase (Novozymes 50010, 42 CBU/g-cellulose, Novozymes A/S, Bagsvaerd, Denmark), two kinds of antibiotic powder;  40 μg tetracycline (LKT Laboratories, Inc., MN, US) and 30 μg cycloheximide (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 10 mL of water were added. The solution was incubated at 50 ºC for 72 hours in an incubator (Sanki Seiki Co., Ltd., Osaka, Japan), and the glucose content of the solution was then measured by liquid chromatography (LaChrom Elite L-2490 Refractive Index (RI) detector, LaChrom Elite L-2200 Auto sampler, LaChrom Elite L-2130 Pump, LaChrom Elite L-2350 Column oven, Hitachi High-Technologies Corporation, Tokyo, Japan).

4. Testing results

4.1 Results of the Component Analysis and Saccharification Testing

The results of the component analysis and saccharification testing of the KSE-processed samples are as follow:

Corn stover:

Component analysis: glucan 45.0%, xylan 20.9%, galactan 0.0%, arabinan 2.6%, lignin  19.4%, ash 1.9%, and extractives 14.3% 

Saccharification testing: The saccharification efficiency was 88.9%. Little or no glucose was eluted when a blank solution (sample only, without enzymes) was used.

Corn dust:

Component analysis: glucan 64.2%, xylan 5.6%, galactan 1.3%, arabinan 3.2%, mannan 0.0%, lignin 7.2%, ash 1.3%, and extractives 15.2% 

Saccharification testing: The saccharification efficiency was 100%. Little or no glucose was eluted when a blank solution (sample only, without enzymes) was used.

Broken corn:

Component analysis: glucan 70.7%, xylan 2.1%, galactan 0.4%, arabinan 1.9%, mannan  0.0%, lignin 6.0%, ash 0.7%, and extractives 13.2% 

Saccharification testing: The saccharification efficiency was 100%. Little or no glucose was eluted when a blank solution (sample only, without enzymes) was used.

4.2 Influence of Heat Treatment Time on Saccharification Efficiency

4.3 Fermentation Experiment

A fermentation experiment was conducted to observe the fermentation of the KSE-processed samples. In this experiment, KSE-processed corn stover (100 g, Fig. 4 Panel A, middle), malted rice (http://en.wikipedia.org/wiki/Jiuqu; for utilization of the hydrolytic enzyme within; 2 g, Maruai Shimizu Brewer, Hokkaido, Japan), baker's yeast (0.3 g, instant dry yeast containing 1.5% sorbitan fatty acid ester, S. I. Lesaffre, France), and water (0.5 L, 23 oC) were placed in a 1.5 L polyethylene terephthalate container fitted with a lid, and left at room temperature (23 oC). After three hours in the container, initial venting was carried out as the container had swelled due to increased internal pressure. Additional venting was carried out four times at two-hour intervals. A different type of dry yeast produced by Nippon Beet Sugar Manufacturing Co., Ltd. (Shimizu Bio Factory, Hokkaido, Japan) was used and a second experiment was conducted. The results of the second fermentation test were very similar to those of the initial experiment. An additional fermentation experiment using the corn stover samples, milled to form a fine powder of 105 μm in size using a pin mill (Nara Machinery Co., Ltd., Tokyo, Japan), without application of the KSE method was conducted. The same malted rice (2g, Maruai Shimizu Brewer, Hokkaido, Japan) and baker’s yeast (0.3 g, instant dry yeast containing 1.5% sorbitan fatty acid ester, S. I. Lesaffre, France) were added to the unprocessed corn stover powder (100 g) of 105 μm in size. As expected, no fermentation was observed.

4.4 Fermentation Residue

Following the fermentation experiment described above in 4.3, the residue left after the collection of the ethanol was washed and dried. This residue (Fig.5) is considered to be mainly composed of lignin and free of the chemicals conventionally used to extract lignin.

5. Discussion

As the KSE method can be applied to first generation technology using grains such as whole-grain corn, and as both the starch and carbohydrate contents can be simultaneously saccharified after application of the KSE method, the cost is substantially lower than that associated with existing technology. In addition, broken corn and corn dust, which are removed from the raw material when first generation technology is applied, can be used. 

The principle underlying the application of the KSE method to herbaceous biomass can also be applied to ligneous biomass. However, the core element of the apparatus shown in this paper is not optimized for the processing of such ligneous material and has not yet achieved a high saccharification efficiency. The current saccharification of ligneous biomass using the KSE method is 20.8% according to the results of saccharification testing conducted by Hokkaido Research Organization in March, 2014, and further research is currently underway.

100% saccharification of materials processed by the KSE method has been achieved. The glucose content was measured by NREL protocol. However, as a 100% saccharification rate is unbelievably high, it is speculated that some other saccharide substances other than glucan, such as xylan, underwent simultaneous saccharification. Further efforts are required in the future to clarify this point.

The fermentation experiments revealed that using the KSE method allowed the rapid induction of saccharification and fermentation on the addition of commonly available enzymes and yeast; however, other enzymes and yeast that are able to improve efficiency levels may exist. Further work on selecting efficient enzymes and yeasts should be entrusted to specialists in the field.

Lignin has been the focus of a great deal of research; however, the extraction of lignin without the use of chemicals such as sulfuric acid has not been achieved to date. The ability to extract lignin without using chemicals shown above may lead to acceleration in research on this material. Further study of this finding needs to be entrusted to the relevant specialists.

6. Conclusions

The research revealed the followings: 1) through the application of …, the saccharide content in lignocellulose biomass can be exposed rapidly, 2) once the saccharide content is exposed, saccharification and fermentation are immediately induced by adding enzymes, yeast and water, and 3) even commonly available enzymes and yeast are sufficient once the saccharide content is exposed. Therefore KSE method enables a large reduction in production costs of ethanol from lignocellulose biomass.

Acknowledgements:

References:

Fig. 4:

Raw materials, KSE-processed materials and pulverized KSE-processed materials;
(Panel A) From left to right: raw corn stover as received, KSE-processed corn stover, and pulverized KSE-processed corn stover. (Panel B) From left to right: raw corn dust as received, KSE-processed corn dust, and pulverized KSE-processed corn dust. (Panel C) From left to right: raw broken corn kernels as received, KSE-processed broken corn kernels, and pulverized KSE-processed broken corn kernels.

Fig. 5:

Photograph of the washed and dried residue left after the collection of the ethanol


Component Analysis and Saccharification Test of Corn Dust and Broken Corn

1.Test samples:

Corn dust (Grain dust) and broken corn specially processed by Kato Method

2. Components analysis

(1) Analytical method

 The analysis was conducted in accordance with the Standard Biomass Analytical Procedures developed by NREL. The extractives, saccharides, lignin and ash were analyzed.
 1) Extractives: NREL/TP-510-42619
Materials extracted by water and alcohol
 2) Saccharides and lignin: NREL/TP-510-42618
 3) Ash: NREL/TP-510-42622

(2) Analytical result

The ratios of glucan within these samples were between 64% and 71%, and a little of xylan, galactan, and arabinan were contained within them.  Though the content of glucan is the sum of cellulose and protein, the content ratio was not revealed because the quantity of the protein was not measured.

Chart1. Components Analytical Result
ExtractivesGlucanXyranGalactanArabinanMannanLigninAshTotal
Corn dust15.2%64.2%5.6%1.3%3.2%0.0%7.2%1.3%98.0%
Broken corn13.2%70.7%2.1%0.4%1.9%0.0%6.0%0.7%94.9%
Chart1. Components Analytical Result

3. Saccharification test

(1) Test method

The saccharification test was conducted in accordance with NREL/TP510-42629;

  • - Each sample weighted 0.1 g of cellulose was added to 30 mL glass scintillation vials.
  • - To each vial, 5.0mL sodium citrate buffer, cellulase (Novozymes 50013) 25FPU/g-cellulose,     β-glucosidase (Novozymes 50010) 42CBU/g-cellulose and antibiotic were added.
  • - 10.00 mL distilled water was added to each vial for increasing the necessary cubic content.
  • - Each vial was cultured in the incubator for 72 hours at 50℃.
  • - The glucose concentration was measured by liquid chromatography (HPLC).

(2) Test result

  • - The saccharification efficiencies of both corn dust and broken corn were 100%.
  • - Without the added enzyme, the glucose yields were almost zero.

(Contact Info)

Toshihiro Kitaguchi
Hokkaido Research Organization/ Industrial Research Institute
Kita19-jo, Nishi11-chome, Kita-ku
Sapporo-shi, Hokkaido 060-0819
Tel: +81-11-747-2949, Fax: +81-11-726-4057
Email: kitaguchi-toshihiro@hro.or.jp
HP: http://www.iri.hro.or.jp/


Saccharification Test of Corn Stover

1. Test samples:

Corn stover: Pioneer Hybrid Japan 39B29, from Shintoku-cho, Hokkaido
1) I, Toshihiro Kitaguchi, milled the corn sover by cutter-mill machine, and classified into mesh size between 20 and 80.  Then the samples were handed to Mr. Kato.
2) Then the samples were specially processed by Kato Method.

Chart1. Components Analytical Result of Corn Stover
 GlucanXyranGalactanArabinanLigninAshProteinExtractives
Acid-
insoluble

Acid-
soluble
Lignin
Pioneer
39B29
45.0%20.9%0.0%2.6%18.2%1.3%19.4%1.9%1.3%14.3%

2. Saccharification test

(1) Test method

The saccharification test was conducted in accordance with NREL/TP510-42629;

  • - Each sample weighted 0.1 g of cellulose was added to 30 mL glass scintillation vials.
  • - To each vial, added 5.0mL sodium citrate buffer, cellulase (Novozymes 50013) 25FPU/g-cellulose,     β-glucosidase (Novozymes 50010) 42CBU/g-cellulose and antibiotic were added.
  • - 10.00 mL distilled water was added to each vial for increasing the necessary cubic content.
  • - Each vial was cultured in the incubator for 72 hours at 50℃.
  • - The glucose concentration was measured by liquid chromatography (HPLC).

(2) Test result

  • - The saccharification efficiency of corn stover was 88.9%.
  • - Without the added enzyme, the glucose yields were almost zero.

(Contact Info)

Toshihiro Kitaguchi
Hokkaido Research Organization/ Industrial Research Institute
Kita19-jo, Nishi11-chome, Kita-ku
Sapporo-shi, Hokkaido 060-0819
Tel: +81-11-747-2949, Fax: +81-11-726-4057
Email: kitaguchi-toshihiro@hro.or.jp
HP: http://www.iri.hro.or.jp/