PROCEEDINGS OF SUGARCANE 2023

149 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING 4.4 The V1 vapour consistency (pressure, temperature and available flow rate) was achieved which improved the process house operations, and specifically a reduction in refinery Vacuum Pan cycle times thus providing increased capacity to the Refinery, 4.5 The sizing of the MVR system was based on the milling rate of 8,000 TCD. As milling rates above 7,500 TCD were not achieved there was an imbalance of the V1 vapour & exhaust steam generation requiring the MVR’s to be operated below their rated capacity to prevent discharge of excess exhaust steam. Sutech will, at a future date, be sending a team to be on-site to fully process commission the MVR installation together with the Factory staff and work towards achieving the initial objective of the study. The results of this revisit will be the topic of Part 2 of this paper. 5. ACKNOWLEDGMENT The authors would like to express appreciation for the support of the Filipino Factory Staff & Management 6. REFERENCES 1Rein, Prof. P.W. – Cane Sugar Engineering, 2007 - ISBN 978-3-87040-110-8

150 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING ABSTRACT: This technical paper looks at ways of achieving, high pol extraction performance from a milling tandem, lowering bagasse moistures and improving the mechanical efficiency of milling tandems. To achieve these results, we need to focus on - very fine cane preparation, high maceration efficiency, well set and maintained mills, high performance evaporator set - high imbibition % fibre, preferably using 6 roll mills as well as having skilled operators and Staff. My yearly averages for a milling season - • First mill - 48% bagasse moisture and 84% pol extraction, • Final, 4th mill - 40% bagasse moisture and 98% pol extraction • 345% imbibition on fibre due to lots of steam, from low bagasse moisture. All checked and verified by Australian Sugar Research Institute Staff for the Australian Sugar Industry. Cane preparation. - The cane shredder is the most important machine in the factory. 90% POC / PI is nowhere high enough for high milling tandem extraction performance (10% juice cells unopened) The Kimberley cane preparation system greatly improves milling performance. KIMBERLEY SHREDDER CANE PREPARATION Australian Sugar Research Institute (SRI) found, contrary to popular belief, the hardest to open juice cells have the highest juice purity. Unopened juice cells in final bagasse inflates the bagasse pol plus bagasse moisture and reduces extraction performance. I prefer to take the POC sample after the final mill to give the correct preparation performance. Typically, results are from 65% to 75%. So, 25% to 35% of the final pol is still in unopened juice cells. These results prove that the initial cane preparation must be of a very high level. O-041 Requirements for a High Pol Extraction Milling Tandem

151 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Fineness is the best way to check the degree of cane preparation. As the preparation gets finer, >93 POC, the POC result starts to decline. I assume there are juice cells in the small bagacillo particles which decreases the POC figure. There are lots of bagacillo particles in very finely prepared cane. See the above photo. Kimberley Company has a patented Cane Preparation System. The load on the shredder drive is constant unlike other shredding systems, and there is no windage as for other systems, with their loss of fine droplets of cane juice. The cane milling rate is set using this Cane Preparation System. The degree of cane preparation is better than from any other design. And steel objects in the cane supply are caught before the cane enters the shredder. A highly recommended system. Value of a high-performance maceration system - Maceration mixing efficiency is the degree of mixing of the juice in the bagasse exiting the mill with the maceration liquid. If you could obtain 100% mixing efficiency, only 3 mills would be required, each with 50% bagasse moisture, to achieve 98% pol extraction. Macerating mixing efficiency is so important if you wish to obtain high extraction performance, almost as important as the cane shredder. One of the reasons you cannot obtain high maceration efficiency, is the degree of cane preparation. Lots of juice still in unopened juice cells does not mix with the maceration fluid. Another reason is the volume of maceration is insufficient to fully wet all of the bagasse. Better maceration mixing efficiency is why diffusers achieve higher pol extraction compared to milling tandems. Typically milling tandems achieve only 30 to 35% maceration mixing efficiency. The Kimberley patented maceration system has much higher maceration mixing efficiency. Getting close to 100% mixing efficiency depending on the degree of cane preparation. It is so important to have the highest degree of cane preparation as maceration liquid cannot quickly mix with juice inside juice cells. I believe that, four x six roll mills with Kimberley Maceration Systems in front of each mill, plus a Kimberley Cane Preparation System, will achieve higher pol extraction performance than a diffuser tandem, plus have lower bagasse moistures than a diffuser tandem. Ideas to improve the way a mill should work - Bagasse does not expand much after compression in the feed roll nip, only up to about 30% for early mills and less than 20% for final mills. So, the trash plate should be set as high as possible. Never set low. The bagasse compaction above a high set trash plate is lower than on a low trash plate and the trash plate wear is less. Do not curve the trash plate teeth downward, as downward sloping teeth compact bagasse in the feed roller grooves, resisting feeding and wearing the trash plate plus the roller grooves. A high trash plate will generally allow the compacted bagasse to self-feed into the discharge roll nip without force feeding.

152 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING A low trash plate requires force feeding into the discharge roll nip which greatly compacts the bagasse on a low trash plate, causing greater wear compared to a high set trash plate. The wear on the leading face of low set trash plates shows how the top of the trash plate should be shaped. Correct shaped and set trash plates hardly wear. I never ordered new trash plates. Continued to use existing trash plates with weld repairs. Especially on the sides of trash plate teeth, using stainless steel wire and a MIG welder. To save adding hard face welding on the tops of trash plates, I used DUA WEAR PLATE sections that we plug welded to the tops of new or existing trash plates. These wear plates are supplied curved to suit, and the correct length and width of the trash plate, with holes for plug welding onto the top of trash plates. They are easy to install and remove, and they last many milling seasons before replacement is required. Only the tops of trash plate teeth have to be welded. Pitch and angle of mill roller grooves. Large pitch roll grooves will not work as well as small pitch grooved rollers. With large pitch roll grooves, the trash plate must be set lower than for rolls with smaller pitch grooves, which affects feeding into the discharge roller due to the higher angle of nip. Small pitch roller trash plates have a lower angle of nip into the discharge roller so they feed better without high level force feeding required. Smaller pitch groove rollers have more top of groove spot welding so they grip the feed better, and they have more bottom of groove juice escape passages. As stated above, the Kimberley patented Cane Preparation System collects the metal objects than enters the milling tandem with the cane supply, before the cane shredder, so these metal pieces do not cause problems breaking roll grooves, so wide angle grooves are not required. Wide groove angles (50 degrees) cannot work without costly lotus drilled rolls as they have bagasse compacted hard against the bottom of the top roll grooves in the feed roll nip, preventing juice the discharge roller compaction has extracted, from moving forward and out of the feed roll nip, Instead, the juice passes out with the bagasse inflating the bagasse moisture. I tried a top roller drilled for half the length of the roller and the full length of the roller with narrow angle grooves. Compared bagasse samples from both ends of the roller. There was no benefit with the drilled section. Drilled rollers are very costly, and the drilled holes plug up with bagasse and no longer work. The feed roll compaction must not fill the top roll grooves,

153 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING So, the top roll groove angle should be low enough, with welding on the sides of the grooves, (2 to 3mm diameter weld lumps) to stop the grooves from filling completely. However, the feed roll nip compaction should be as high as possible to obtain high extraction performance. The tops and bottoms of roll grooves should not be greater than about 3mm wide. Most of the roll pressure is on the tops of roll grooves. Narrow roll tips then cut the bagasse fibres to improve extraction performance, and better fill the grooves. Wide tops of rolls support the bagasse and prevent complete filling of grooves in the top and discharge roll nips. Narrow groove bottoms allow juice to escape better than wide groove bottoms. Roll surface speeds The bagasse passing through a mill rotates around the top roll centre. So, the outer surface of the bagasse in contact with the underfeed (fourth) roll, feed roll and discharge roll hasfurther to travel compared to bagasse in contact with the surface of the top roll, So, the bagasse in contact with the outer rollers should move faster than the surface speed of the top roll. The various speeds are illustrated below. Often the diameter of the top roll islarger than the diameters of the outer three rollers. So, it isimportant to have a range of pinions with varying tooth numbers. I used to have a range from 17 teeth up to 21 teeth. This pinion range of tooth numbers allows for setting the three outer rollers at close to the recommended speeds. If the outer roll speeds are too slow, the top roll must try to drive the bottom rollers through the compacted bagasse mat. And the speed of bagasse passing through the mill is retarded on the outer surface compared to the inner surface against the top roll.

154 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING The outer rollers, underfeed roll, feed roll, discharge roll must move at their correct speeds relative to the centre of the top roll. The outer surface of bagasse passing through the mill has much further to travel compared to the bagasse on the inner surface, in contact with the top roller. The setting of the vertical hopper and the underfeed (fourth) roll are very important mill settings. The pressure feeder and the feed roll achieve most of the mill extraction performance. The mill ratio should be as low as possible Typically 1.6 (first mill) to 1.3 (last mill) for 6 roll mills and 1.8 (first mill) to 1.5 (last mill) for 4 roll mills, depending on the angles of roll grooves. Provided the mill is well set and has fine cane preparation, the mill feeding will be good irrespective of the quantity of juice in the bagasse feed. The mill ratio is governed by the groove angle of the top roll plus the amount of welding on the sides of the grooves. The feed roll compaction must not fill the top roll grooves, even though it must be as high as possible. But the discharge roll compaction must fill the top roll grooves. The unfilled space at the bottom of the top roll grooves allows the juice expressed by the delivery roll to escape forward. Not backward with the expelled bagasse. You can check if there is a juice space at the bottom of the top roll grooves by using a with 1.5mm or 2.5mm diameter boudin cable (flexible wire) hung from an overhead crane. This cable should remain slack until it almost reaches the delivery nip.

155 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING During such a test at one sugar factory. the boudin cable passed out beyond the discharge roller with no drag on the cable. The mill Engineer could nor believe what he was seeing. The problem was due to very wide tops of roll grooves which supported the bagasse, preventing the top roll grooves from filling at the discharge roll nip. • Some types of mill cheeks can allow the pintle end of a discharge roller to move away from the cap adjusting bolt if they are not restrained and the mill is running empty. I believe it is wise to jack the pintle ends of the feed and discharge rollers apart so they are hard against the cap jacking bolts before checking settings. The load on the pinion teeth sometimes moves the discharge roller pintle end in towards the feed roll so you can have incorrect settings which can give poor milling results • When installing a mill, it is important to make sure the roll centres each end are equal. If the centres are not equal, the pinion meshing teeth will have loading at one end, so there is end thrust by the pinions on the sides of the roller bronze bearings, causing wear on the ends of bearings plus the sides of trash plate and scraper teeth. Worn sides of bronze bearings should be repaired every maintenance period. • The Kimberley Mill and Pressure Feeder rollers have seals each end of the bearings so a large volume of cooled lubricant can be delivered to each bearing, with the surplus returning to the lubricant storage tank. Induvial small gear pumps deliver the lubricant to each bearing. So, this lubricant system is very reliable. Unlike existing lubricant syatems. • It is important to have easy to use “feeler gauges” for checking and adjusting mill settings. I used to use light easy to use timber feeler gauges covered with thin tinplate which was glued to the timber to prevent wear on the timber by rough roller grooves. These light weight feeler gauges should be available in many different accurate thicknesses. For thin feeler gauges, metal ones are ok. A group of these feeler gauges can be used for checking large roll settings, • Maceration delivery weirs give a more even spread of liquid across the width of conveyors compared to maceration sprays. Keep the maceration liquid off the conveyor chains, if possible, as the maceration liquid promotes faster chain wear. • A discharge roll compaction of 850 kgs / M3. should give moisture of 33.66%. (Solid bagasse weighs 1530 kgs / M3. Water weighs 1000 kgs / M3. 850 kgs / M3 has an equivalent volume of 1530 – 850 = 660 kgs of bagasse or 660 / 1530 x 1000 = 431.37 kgs of water to 850 kgs of bagasse. So, bagasse moisture = 431.37 / (850 + 431.37) x 100 = 33.66%). The difference between the calculated moisture and the actual moisture equates to the amount of hydraulic lift above the calculated lift. This increase in hydraulic lift will give a very close estimation of actual bagasse moisture. (I have checked this) The extra lift is all liquid that has not been extracted from the highly compacted bagasse. Because of the speed of the rollers the liquid does not have time to be expressed fully from the compacted bagasse, especially the juice in the discharge roll nip. So, a very large final mill operating with slow speed rollers will give much lower bagasse moistures than smaller rollers with high roll surface speeds.

156 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Pressure Feeders - 6 roll mills will outperform 4 roll mills. The pressure feeder rollers remove much of the juice before the compacted bagasse enters the feed roll nip, reducing the volume of juice the feed roll has to express. Most pressure feeder chutes are way too long. Generally close to or above 2 meters in length. They must be tapered larger to reduce friction in the chute as they extend from the pressure feeder rollers. About 75mm per metre. So, the wide compacted bagasse in a long chute must be force fed into the feed roll nip. The Kimberley design Pressure Feeder has a very short chute resulting in a much narrower feed at the mill feed roll contact point compared to long chutes, So, often the compacted bagasse in the Kimberley PF chutes self feeds into the mill feed nip without having to force feed the mill. If the thickness of bagasse exiting the chute is lower than about 32 degrees nip angle the bagasse will self feed into the roller nip area. So, a very short pressure feeder chute and high trash plates are required to allow a mill perform well. Cutting scraper plates – Scraper and trash plate teeth should have clearance for the roll welding so it does not get broken off during roll welding. Therefore, it is necessary to find a different way for cutting teeth that will give this clearance. For many years my mill scraper plates have been cut on a special machine that we designed and manufactured. This machine consists of a sliding table on a stand about waste height, The steel section for the scraper plate sits on this sliding table. The table is moved by turning a long-threaded shaft with a pitch to suit the measurement of the roller groove width. Turn the threaded shaft several turns to get the correct roll groove pitch. This table is set beside an oxy acetylene profile cutting machine. We have a special guide (jig) to suit the required tooth shape. The tooth profile can be any shape when using these machines. Initially a travelling oxy acetylene cutting machine, on a track, is used to cut the correct tooth tip angle the full length of the plate. Then the teeth are cut using the special shaped jig on the profile cutting machine. The cutting tip is set at the same angle as the sloping leading edge of the scraper plate. To allow for expansion effects we turn the threaded rod more than normal every (about) 300mm of plate length so the pitch is increased by about 1mm. However, the pitch of the threaded rod could be made to suit this expansion effect. A suitable chain could also be made to work instead of the threaded rod. Cutting a scraper plate only takes about 8 hrs. Faster if multi head oxy acetylene cutters are used. When the teeth are worn, new teeth can be cut on this same plate until its width becomes too narrow, or an extra piece of plate can be welded on to bring the plate back to its original width. Trash plate teeth can be cut the same way. This plate cutting method allows for having a tooth shape that is clear of the spot welding spheres on the roller grooves. KTC manufactures these machines. Imbibition Water Supply - The evaporatorset must work well to have high extraction performance.Ahigh-performance evaporator set will provide a higher volume of imbibition water for the milling tandem, improving its extraction performance, provided the cane has been finely prepared. Things to check with the evaporator set -The set is always working at maximum capacity, controlled by the quantity of imbibition water added to the milling tandem, and the level of juice in the ESJ tank. (Clear juice tank)

157 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Aim to only add water to the tandem rather than to the effet juice supply tank. (ESJ tank) The entrainment arrestors are kept clean Tubes are kept clean. Always check the lower tube section for heavy scale build-up that you cannot see from the top of the calandria. Even after cleaning there can still be a heavy scale build up in the lower (about) 400mm of tubes. Suitable sized downcomer tubesto increase juice circulation. I am a believer of having the maximum amount of juice circulation in vessels. Even adding outside vessel downcomers around vessel calandrias. Maximum vapour bleeding. High feed in juice temperature to first effect. Good juice level control. Operate juice levels as low as possible while fully covering the top of the calandria. Check pressures in effects calandrias and vessel juice spaces often with accurate gauges. These pressures give you the resistance to vapour flows in vapour lines, and through clogging entrainment arrestors. Vapour flows travel at very high speeds so right-angle bends and valves in the vapour lines will increase vapour pressure in preceding vessels and reduce their boiling capacity. The pressure difference between the upstream effect and the downstream vessel calandria should be the lowest possible difference. If high, the capacity of the evaporator vessel will be affected. Having these pressures available, especially when the vessels have clean tubes and are working at maximum capacity, will allow us to improve the factory steam efficiency if required. The delivery end of the evaporator clear juice tank water make-up supply line should be visible and any time the water valve is opened it should be recorded. Especially for an automated makeup feed valve. If the ESJ tank is running low the operator should have the imbibition rate increased to achieve a higher operating tank level. Check the condensate level in the vessel calandrias is low. If a vessel is slightly shaking and noisy, there is a high level of condensate in the calandria. Check the noxious gas pipes are still in good condition. Not rusted off inside the calandria. The inside top and inside bottom of calandrias must be vented. Not the centre section. Check all water lines to juice lines are not leaking. I use T piece connections. Three valves. The centre valve would normally be open to indicate any leaky water/juice valve. Operate with the lowest final vessel pressure possible. Check the inlet feed lines. They should be around the outside of the calandria with the feed out line from the calandria centre. Often, I see very poor inlet feed positions that will greatly affect the performance of the vessels. Some inlet feed lines are installed very close to outlet pipes. I always check the positions of feed in and feed out lines on evaporator vessels in mills I visit. I believe it is a good idea to have juice samples taken from the top of calandrias, as well as from the juice outlet pipe. In a No 1 vessel in my Australian factory, I found a difference of 16 units of brix. 16 units higher above the calandria compared to the juice outflow to the second effect. (Poor juice circulation due to no centre well) I then installed pipe downcomers around this vessel to reduce this brix difference. If a single effect has multi vessels that are operating in parallel there is a chance that one vessel can be “loafing”. Its juice/syrup brix may be higher than in the other effect vessel/s. So, the capacity of the set is reduced. (Lower rate of imbibition for mills) I prefer to have multiple effect vessels in series rather in parallel. So, these vessels must share the “work load” Kimberley can check the performance of an evaporator set if required. Bagasse sampling – Bagasse sampling must give true, accurate results. If sampling from platforms above the mill, you will need a suitable fork device for collecting the samples. It will hold the full sample due to the inward sloping tynes.

158 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING And you will need a clean dry tray for mixing the collected samples on. Example, the top of a 200 litre grease drum is suitable. Full depth samples should be taken a quarter way across the width of the bagasse exiting the mill, mid-way and three quarters the way across. At least 6 samples should be taken. Thoughly mix the samples on the (clean dry) tray then take a sealed sub sample for the lab, and dump the remainder. If the mill normally works with chute height control and runs constantly under load, samples should only be taken when the mill is working with a normal bagasse level in the feed hopper. Take about 6 lab samples per shift. Not less than four samples. A Staff member must often check the samples are taken as described above. Skilled operators and Staff – It is so important for Factory Staff to know how to get and maintain high milling tandem performance. KTC provides week long lecturers for Staff of sugar factories to improve their technical knowledge and skills.

159 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Sombat Khawprateep1 *, Thawatchai Koedsuk1 , Somwang Leekar1 , Wanwanat Chainarong1 and Sanon Boonmee1 1 Farm Mechanics Department, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus. *Correspondence to: [email protected] ABSTRACT: Sugarcane cut by the chopper harvester is as billet supply delivered to the sugarcane mill. The cane billet causes weight loss differently under varying billet quality and time for crushing. Delivering sugarcane billet in available time can improve weight loss and juice destruction. The objective of this preliminary study was to study the weight loss of billet quality after cutting by a chopper harvester. A John Deere 3520 chopper harvester was operated to cut Khon Kaen 3 cultivar (age 10 months) to study the weight loss. Approximately 20–25 kg (5 samples) of billets were tested to evaluate the loss. The billet supply was inspected for the quality of the sugar cane in 3 categories: sound, damaged and mutilated billets. The weight of cane samples was assessed at different intervals every 6 hrs for a total of 48 hrs by testing in the shade. Each category was performed by statistical analysis with a significance level of 0.05. After 48 hrs, the weight of the sound, damaged and mutilated billets reduced by 9.54%, 13.37% and 27.5% respectively. The sound billets were not significantly different (P-value ≤ 0.05). The damaged billets were significantly different (P-value ≤ 0.05) after 48 hers. Additionally, the mutilated billet, after 12 hrs, was significantly different (P-value ≤ 0.05) by reducing 12.32%. From the preliminary data of the billet quality, it is hypothesized that sugarcane billets should be delivered for crushing within 6 hrs or less to reduce the weight loss, especially as the mutilated billet. This often occurs in older chopper harvesters where the tip speed ratio is not optimal between feed-trained rollers and chopper system. Keywords: chopper harvester, sugarcane loss, billet quality P-013 Preliminary study of weight loss via billet quality cut by a chopper harvester

160 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Akeme Cyril Njume, Yumika Naomasa, Yoshinari Izumikawa, Kittipon Aparatana, and Eizo Taira* Department of Regional Agricultural Engineering, University of the Ryukyus, Okinawa, 903-0213, Japan *Correspondence to: Department of Regional Agricultural Engineering, University of the Ryukyus, Okinawa, 903-0213, Japan. E-mail: [email protected] ABSTRACT: There are largely two methods of extracting sugarcane juice in raw sugar factories in Japan. The two squeezing methods are direct squeezing of the cane stalk immediately after the removal of trash and cleaning, and the shredding of the cane into convenient sizes before squeezing with a hydraulic press machine. The present study investigates the effect of the two squeezing methods on the NIR measurement of pol in juice through the development of calibration model. A benchtop NIR spectrometer of wavelength from 400 to 2500 nm was used for spectra acquisition of 424 samples of different varieties. Spectra data were pre-processed with standard normal variate (SNV), and derivatives: first and second with 21 segments. Partial least square regression (PLSR) was deployed for model development by using 8 PLS factors. We found that the mean shredded-squeezed spectrum had higher absorption compared to the direct squeezing spectrum with a similar orientation. The best calibration model for commercial pol in juice was obtained from the SNV pre-processing method of the shredded and squeezed method with performance accuracy of 0.99, 0.20, 0.973, 0.27, and 6.12 for R2c, RMSEC, R2p, and RPD, respectively. Meanwhile, the best model for directing pressing was obtained from the second derivative with performance accuracy of 0.924, 0.61, 0.883, 0.79, and 2.81 for R2c, RMSEC, R2p, RMSEP, and RPD, respectively. Based on our results, NIR measurements of pol in juice in sugarcane factories could be accurately measured when samples are shredded before being squeezed. Keywords: calibration model, NIR measurement, pol in juice, squeezing methods, and sugarcane factories P-038 Investigating the influence of squeezing methods on NIR spectrometer measurement of pol in juice

161 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING ABSTRACT: When choosing the primary decolorizing process in a cane sugar refinery, the processor has 3 practical options: 1. Regenerable ion exchange resin (IER) 2. Regenerable granular activated carbon (GAC), 3. Non-regenerable powder adsorbents, including powder activated carbon (PAC), high performance adsorbents, and multi-functional adsorbents Each of these has advantages and drawbacks, including capital requirements, local energy costs, environmental issues, and other factors. The use of the new generation, high-capacity multi-functional adsorbent with superior filtration properties, Ecosorb® S-451, allows efficient re-use in situ, resulting in a simplified operation compared to other approaches to re-using powder adsorbents. A properly designed re-use system will reduce adsorbent consumption per ton of refined sugar by 30 – 40% compared to single use, making it economically attractive compared to the two regenerable column approaches. Additional benefits include the ability to run at higher brix (up to 70 brix), resulting in significant energy savings, and the ability to instantly respond to changes in raw sugar color, allowing a consistent color feeding the crystallization process. S-001 Ecosorb® Multi-functional Adsorbent with Re-Use as Primary Decolorizing Process in Cane Sugar Refining

162 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Nakul Phase1*, Mohan Gawade2, and Pravin Waghmareme3 1Praj Industries Limited, Pune, 411057, India 2Praj Industries Limited, Pune, 411057, India 3Praj Industries Limited, Pune, 411057, India *Correspondence to: MFPE Department, Praj Industries Limited, 274 & 275/2 Praj Tower, Hinjewadi Road, Hinjewadi, Pune, 411057, India, [email protected] ABSTRACT: Two undesirable carbohydrate polymers in sugar production, dextrans and Starch give rise to challenges like increased viscosity of streams, reduced sugar recovery and the quality of sugar produced. Knowing these problems, Praj has developed a unique blend of enzymes named “JUICEZYME” that effectively breaks down the polymers resulting in enhanced Process efficiency, Sugar Yield and Sugar quality. Application Of Juicezyme @ 2.0 to 3.0 ppm in Mix Juice / Syrup has been demonstrated in sugar factories in India, SEA and LATAM regions. With application of Juicezyme, effective degradation of the dextrans and starch was observed. Juicezyme application was carried following an optimized dosing and analysis protocol under sugar factory standard conditions wherein, the dextran and starch contents in different streams from Juice to final sugar were analyzed for 2 to 3 days before and during Juicezyme application and monitored every 24 hrs. For this, representative samples of Juice, Syrup, Massecuites and Molasses were taken and analyzed for Dextran and Starch by ICUMSA modified alcohol haze method. Within 72 hrs. of Juicezyme application, 70 to 80% of dextran and starch were hydrolyzed. The final Sugar ICUMSA colour reduced below 100 IU. In the final white sugar, the dextran and starch content reduced below 100 ppm which showed improved sugar quality for specific beverage applications. A significant increase in sugar recovery by 0.2 to 0.5% was observed. Keywords: Dextran, Starch, Juicezyme, ICUMSA S-002 Demonstration of “Juicezyme” Application and Benefits for Sugar Industry

163 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING 1. INTRODUCTION: From several challenges that impact the production of sugar from sugarcane, the challenge of two carbohydrate polymers – Dextran and Starch (polymers of glucose) directly affect the production process and the quality of sugar produced. The major factor contributing to sugar cane deterioration and therefore the sugar recovery and quality are microbial infections (1). The microbial infection of sugarcane, and the extracted juice is affected by Climate temperature, humidity, Mechanical- Cutting, chopping, Time from farm to mill (2). The infecting bacteria rapidly consume sugar and enzymatically convert to dextrans. While dextrans are generated by microbial attack, mainly the lactic acid bacterium Leuconostoc mesenteroidis [3], right from stage of harvesting, the starch has a natural presence in sugarcane as product of photosynthesis (4). The microbial attack happens due to exposure of internal tissues of sugar cane during cutting, chopping, or burning (1). During cane crushing and juice extraction, both these polymers are extracted in the juice and during processing lead to increase viscosity, inhibit crystal formation, polarization distortion and loss of sucrose to molasses. The high viscosity is associated with high molecular weight portions of dextrans (>1000KDa) which affect the boiling house operations, reducing the rate of evaporation and crystallization (1). Viscosity during clarification reduces precipitation speed of impurities, forming scale deposits and decreases heating efficiency and hence higher energy loss. The massecuite cooking time increases and reduces their exhaustion. The increased crystallization time cools the massecuites and further contributes to increasing viscosity. The purge time of centrifugals goes up to get required sugar quality. The raw sugar from such massecuites is sticky and therefore causes difficulty in handling and packing. The needle shape crystal formation reduces the purging efficiency of the massecuites in the centrifugals resulting in a poor separation of crystal and molasses, hence reducing the refined quality of the sugar (2) (5). These polymers are not completely removed in the processing and end up in to raw / final crystal sugar thus affecting sugar quality. Unlike dextrans, Starch, a primary product of photosynthesis stored in stalks but more abundant in leaves and growing point regions. Starch is found in all sugarcane products including raw and refined sugar and in sugarcane mills and refineries although its concentration varies greatly depending on the season, variety, sugarcane diseases, maturity, processing conditions, and analysis method (4) (6). Like dextran, presence of starch contributes to loss and inefficient production in sugar mills (4). Starch in sugarcane juice interferes in the clarification, filtration, and crystallization processes. During processing, they increase viscosity, inhibit crystallization, and increase the loss of sucrose to molasses. They may also contribute to polarization distortions. It is known that polysaccharides such as starch are not completely extracted during processing, and that they end up being incorporated into raw crystal sugar. Studies have shown that 200-250 ppm starch level in raw sugar refinery may cause problems during processing (5). Praj’s study of sugar mill streams showed that the concentration of dextrans in Juice and syrup ranges between 400 to 1500 ppm, whereas concentration of Starch was found to be about 400 ppm. To investigate the effect of dextran and Starch degradation on sugar production and quality, application studies were conducted using Praj’s ‘Juicezyme’, enzyme – a unique blend of thermostable enzymes. Juicezyme is active over a wide range of pH and Temperature. The optimum Temperature range is 60 to 80 Deg. C whereas pH range is 5.3 to 6.80. Results from one such study in sugar production plant in Philippines are presented here. 2. MATERIALS AD METHODS: Analysis requirementChemicals (Purity - All chemicals of Analytical grade) - Dextran standard (T - 500), Trichloroacetic acid, Absolute alcohol, Sucrose, Acid washed kieselguhr, Acetic acid, Potassium Iodate, Soluble Starch. Glassware - Volumetric Flask – 50 ml, 100 ml, 250 ml and 500 ml, Whatman filter paper No. 1, Whatman filter paper No. 5\, Test Tubes - 50 ml capacity, Auto Pipette 1.0 ml and 10 ml, Measuring Cylinder - 25 ml capacity, Glass Pipettes - 25 ml capacity, Funnels plastic or glass, Beakers - 100 ml, Glass Rods Equipment -Electronic Balance -Capacity 0.1 mg list count UV/VIS Spectrophotometer. Method of Analysis: Modified Alcohol haze method for estimation of dextran and Starch content – Ref: Dextran analysis - (ICUMSA GS1/2/9-15 (2015), Starch analysis – ICUMSA G11-16 (2013): Following are the details – Standard graphs were plotted using Dextran standard in 100 to 1000 ppm range and Starch standard in 50 to 500 ppm range. The dextran and starch content in the analysis samples were calculated using linear equation from standard graphs multiplied with dilution factor.

164 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Juicezyme Application: Method of Application: • Juicezyme is dosed @ 3.0 ppm on per day cane crushing capacity in MT (TCD) of the sugar mill e.g., for 5000 TCD mill, required quantity of Juicezyme is – 5000 TCD X 1000 X 3.0 ppm = 15.0 Kg/day. • The required quantity of Juicezyme is diluted with clean cold water and used for application. The diluted Juicezyme is dosed continuously in clarified syrup. • Following are the details of Juicezyme application monitoring executed in a sugar producing plant in Philippines. The Juicezyme application in the was done in two phases as described in Table below: - Before starting enzyme application, samples were taken from – Syrup, Massecuite A, B, C, Molasses A, B, Final Molasses and Raw Sugar, for two days and analysed for Dextran and Starch content. - Standard analysis methods were followed for analysis of Dextran and Starch – Modified Alcohol Haze method for dextran (ICUMSA standard). - Quantity required of Juicezyme was calculated based on the current running TCD of the sugar mill and added continuously- the mill was running at 6,000 TCD, accordingly the required quantity of Juicezyme @ 3.0 ppm dose was worked out to 18 Kg per day. - The enzyme dosing was done in clarified syrup. Before dosing, the required quantity of enzyme per day was diluted with water – 18 Kg diluted to 600 Liters. - The dilute enzyme solution was dosed continuously to syrup using a dosing pump. - Samples were taken from each stream mentioned above after 24 hrs, 48 hrs and 72 hrs of Juicezyme application and analysed for Dextran, Starch. The raw sugar samples were additionally analyzed for ICUMSA colour. - The required quantity of enzyme was adjusted based on variations in the cane crushing on daily basis. The enzyme dose was maintained at 3 ppm. Results: Dextran and starch content during Juicezyme application were compared with the average values of Dextran and Starch analysed and observed before Juicezyme application and accordingly the percentage reduction was calculated. Following tables shows the data of Dextran (Table-I) and Starch (Table-II) ppm and percentage reduction in Dextran and Starch content in different samples during Juicezyme-D application. The ICUMSA colour data of raw sugar is shown in Table-III. Sugar recovery data shown in Table V.

165 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Table I: Dextran degradation monitoring data – Table II: Starch Degradation monitoring data -

166 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry SUGAR PROCESSING Table III: Sugar ICUMSA Colour of Raw Sugar: Table IV: Sugar Recovery data: Observations / Remarks: • Significant reduction in all the streams for percentage dextran reduction was observed. Dextran’s reduction at 72 hrs. of Juicezyme application was 77 to 82% in different streams. • Despite having a high burnt cane percentage – 30 to 35% during demonstration, which results in high dextran content, Juicezyme has effectively degraded the Dextran. • Significant starch reduction was observed in all the streams – 80 to 90 % in all streams. • Sugar recovery increased by 0.43% in the three days of Juicezyme application. • Improvement in the sugar ICUMSA color was observed. This shows improvement in the quality of sugar. • During application of Juicezyme, viscosity of Massecuite A, B and C was reduced, this resulted in improvement in sugar recovery. The reduced viscosity benefits with reduced requirement of viscosity reducing agent in the mill. • Improvement is seen in the size of sugar crystals. 3. CONCLUSION: It is concluded that Praj’s Juicezyme is highly effective in degrading the undesirable polymers dextran and starch simultaneously within 24 to 72 hrs. of application. The degradation of these polymers resulted in reduced viscosities of the massecuites and thus helped increase the recovery of sugar. 4. REFERENCES: [1] Gillian Eggleston, Adrian Monge, Belisario Montes, David Stewart (2009) Application of Dextranases in sugarcane factory: Overcoming practical challenges, Sugar Tech, 11 (2). Pp 135-141. [2] Efraín Rodríguez Jiménez (2004), The Dextranase along sugar-making industry, Biotechnologia Applicada, Vol. 22, No.1.PP 20-27. [3] Mackrory, L. M., Cazalet, J. S., & Smith, I. A. (1984). A comparison of the microbiological activity associated with milling and cane diffusion. In Proceedings of the 1984 South African Sugar Technologists Technical Association meeting, p. 86–89. [4] Joelise de Alencar Figueira, Priscila Hoffmann Carvalho, Hélia Harumi SATO (2011) Sugarcane starch: quantitative determination and characterization, Ciênc. Tecnol. Aliment., Campinas, 31(3), PP 806-815. [5] Cuddihy, J. A.; Porro, M. E.; Rauh, J. S. (2001) The presence of total polysaccharides in sugar production and methods for reducing their negative effects. Journal of American society of Sugarcane Technologists, v. 21, PP 73-91. [6] IMRIE, F. K. E.; TILBURY, R. H. (1972) Polysaccharides in sugar cane and its products. ugar Technology Reviews, v. 1, PP. 291-361.

167 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS

168 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Piyanan Boonphayak1,2*, and Sirikarn Khansumled1,2 1 Department of Industrial Engineering, Faculty of Engineering, Naresuan University, Phitsanulok, 65000, Thailand 2 Center of Excellence in Biomaterials, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand *Correspondence to: Department of Industrial Engineering, Faculty of Engineering, Naresuan University, Phitsanulok, 65000, Thailand. Email: [email protected] ABSTRACT: Sugar is an essential ingredient in the cooking of people worldwide, and Thailand has sugarcane fields spread out in the north, central, eastern, and northeast parts of the country. Therefore, there is a lot of space to grow sugarcane to feed raw materials to sugar mills. Moreover, growing a lot of sugarcane causes the problem of fine particulate matter (PM2.5) due to the burning of sugarcane leaves to make it easier to harvest. However, burning sugarcane leaves will cause air pollution problems and affect the quality of the sugar in sugarcane. Therefore, this study used the sugarcane leaves as raw material to extract the biosilica for a substitute import via the sol-gel method and its application in biodegradable polymers. The biosilica powder was characterized using X-ray diffraction (XRD), a field emission scanning electron microscope (FESEM) with energy-dispersive X-ray (EDX) analysis, Brunauer-Emmett-Teller (BET), and Fourier transform infrared (FTIR). EDX observed higher amounts of SiO2 particles from the synthesized nanoparticles. Moreover, BET measured the surface area corresponding to 177 m2/g. The results from this study indicated that sugarcane leaves can be used as raw materials to extract biosilica powder. The extracted biosilica powder is reinforced with biodegradable polymer composites based on polymer blends of polylactic acid and thermoplastic starch (PLA/TPS). The effect of different biosilica concentrations (0, 1, 3, and 5 percent by weight). The results demonstrated that an increase in biosilica decreased the mechanical properties of polymer composites due to the agglomeration of nanosized biosilica. This result was supported by the morphology of biodegradable composites as revealed by FESEM, which revealed that biosilica has poor interfacial adhesion. However, the biosilica enhanced the properties of both elongation at break and biodegradability. Significantly, the current research has demonstrated that PLA/TPS/biosilica can be considered for food packaging applications. Keywords: Sugarcane leaves, Biosilica, Biodegradable, Polylactic acid, Thermoplastic starch O-001 Extraction and characterization of biosilica from sugarcane leaves waste and its biodegradable application

169 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Boontiwa Ninchan*, Supawat Songbang and Chanyanuch Noidee Sugars and Derivatives Analytical Laboratory, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900 THAILAND *Correspondence to: Sugars and Derivatives Analytical Laboratory, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900 THAILAND. Email: [email protected] ABSTRACT: Oligofructans are potential biological substances that due to their distinctive properties have a positive health-promoting effect then widely used in many industries. They are fructose-containing molecules joined by beta-glycosidic bonds. Oligofructans can be produced using levansucrase-producing microbial fermentation with a sucrose-based substrate. This research aimed to compare the efficiency of carbon sources at different concentrations by Bacillus subtilis TISTR 001 fermentation and to study the prebiotic properties under in vitro conditions. Three carbon sources(sucrose,sugarcane juice, and molasses) and four substrate concentrations (15, 20, 25, and 30 °Brix) were studied under fermentation at pH 6.8, 30 °C, and 150 rpm for 168 h. The results showed that all substrates could be a suitable carbon source and had the same trends for both the production of levansucrase and oligofructans that were positively correlated with the substrate concentration. Oligofructans formation was confirmed by the changes in the sugars (sucrose, glucose, and fructose) and the produced oligofructans (kestose, nystose, 1-fructofuranosylD-nystose, and levan). Sugarcane juice at 30 °Brix was the best carbon source with the highest levansucrase activity (2.81 x107 Unit/mL) at 48 h and the maximum oligofructans content (87.6 g/L) at 60 h with the kinetic parameters: specific growth rate of 0.12 h-1, substrate consumption of 2.93 g/L/h, production yield of 0.36 g/g reducing sugar, and productivity of 1.10 g/L/h. Moreover, oligofructans had potential functional ingredients that exhibited prebiotic properties to resist the digestion of enzymes in the gastrointestinal tract under in vitro conditions with digestion of only 6.92%. In addition, they acted to promote the growth of prebiotics, especially Bifidobacterium bifidum TISTR 2129 and inhibited the growth of pathogens using both single culturing and co-culturing with probiotics. These results identified the optimum conditions for efficient oligofructans production and clarified prebiotic properties that assist further research on the application of functional ingredients. Keyword: Molasses, Oligofructans, Prebiotic, Sucrose, Sugarcane juice O-017 Production and prebiotic properties of oligofructans by Bacillus subtilis TISTR 001 fermentation as potential functional ingredients

170 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Nisit Watthanasakphuban1*, Boontiwa Ninchan¹, Phitsanu Pinmanee² and Kittipong Rattanaporn¹ 1 Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 10900, Bangkok, Thailand ²Enzyme Technology Research Team, National Center of Genetic Engineering and Biotechnology (BIOTEC), 12120, Pathum Thani, Thailand *Correspondence to: Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 10900, Bangkok, Thailand, E-mail: [email protected] ABSTRACT: D-psicose is an alternative rare sugar that has a high sweetness at about 70% of sucrose but provides very low calories (0.007 kcal/g). D-psicose is easily applied in various food products as a sucrose alternative due to no bitter aftertaste and could improve the egg-white protein foaming properties. D-psicose has been reported an anti-inflammatory properties as well as antioxidant activity, leading to highly interested in the food and functional food industry. The synthesis of D-psicose has been done using D-psicose-3-epimerase enzyme conversion from fructose substrate. Meanwhile, the chemical method which has been used for many sweeteners production cannot be used to synthesize the highly specific form of D-psicose. The limitation of D-psicose production using enzymesfrom wild-type microorganisms exhibited low D-psicose yield, which is not applicable for industrial applications. In this study, recombinant DNA technology has been applied to enhance the D-psicose-3-epimerase enzyme production. The recombinant strain showed about a 10-fold increase in D-psicose-3-epimerase enzyme production compared to the wild-type strain. The purified recombinant D-psicose-3-epimerase enzyme showed about 30% D-psicose conversion when fructose was used as a substrate. This successful increase in D-psicose-3-epimerase enzyme production is a major step for the industrial-scale production of D-psicose in Thailand. Keywords: D-psicose, D-psicose-3-epimerase, rare sugar, enzyme purification O-018 D-psicose synthesis using recombinant D-psicose-3-epimarase enzyme

171 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Ukrit Samaksaman 1, 2*, Thanatsan Poonpaiboonpipat³, Dong Shang¹ and Supanut Pattanasarin⁴ ¹Department of Natural Resources and Environment, Faculty of Agriculture Natural Resources and Environment, Naresuan University, Phitsanulok, 65000,Thailand ²Excellence center of energy environment and smog disaster problem, School of Renewable Energy, Maejo University, Chiangmai, 50290, Thailand ³Department of Agricultural Science, Faculty of Agriculture Natural Resources and Environment, Naresuan University, Phitsanulok, 65000, Thailand, [email protected] ⁴Piriyalai School, Phrae, 54000, Thailand, [email protected], [email protected] *Correspondence to: Department of Natural Resources and Environment, Faculty of Agriculture Natural Resources and Environment, Naresuan University, 99 M9 Thapho, Mueang, Phitsanulok, 65000, Thailand. E-mail [email protected] ABSTRACT: Separate hydrolysis and fermentation (SHF) of sugarcane tops (SCT) for bioethanol production appears viable and economically feasible, especially in view of ongoing global energy demands. Prior the tests, biomass composition and physicochemical properties of SCTs were investigated. In the SHF process, SCTs were pretreated with different hydrolytic substrates such as sodium hydroxide, sulfuric acid, and the α-amylase enzyme before being subjected to alcoholic fermentation using distinct yeasts from both commercial and northern Thai liquor sources. The levels of reducing sugar and alcohol yield were analyzed during the pretreatment and fermentation stages, respectively. The experimental results showed that the reducing sugar contents were in the range of 28.5-40.1 mg/mL, which found higher in hydrolysis with sulfuric acid and enzymatic substrates than in sodium hydroxide substrate. On the fourteenth day of fermentation, diverse yeasts led to the highest bioethanol yield in the case of commercial yeast (4.3-4.9%) while fermentation with a local yeast resulted in a comparatively lower yield. Notably, the SHF processes involving sulfuric acid and α-amylase substrates along with fermentation using commercial yeast, exhibited the most promising potential for SCT conversion into bioethanol. Additionally, for cases involving local yeasts, coupling the SHF process with the α-amylase enzymatic substrate is suggested to enhance efficiency in production. Keyword: Bioethanol, Fermentation, Hydrolysis, Pretreatment, Sugarcane tops, Yeast O-021 Separate hydrolysis and fermentation process of sugarcane tops for bioethanol: Effect of different yeasts from northern Thai liquor and commercial sources

172 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Prapassron Rugthaworn1 *, Udomlak Sukatta¹, Ketsaree Klinsukhon¹, Lalita Khacharat¹, Surisa Sakayaroj¹ and Prakit Sukyai³ 1 Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, Bangkok 10900, Thailand ²Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand ³Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand *Correspondence to: Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, Bangkok 10900, Thailand. E-mail [email protected] ABSTRACT: The aim of this study was to enhance yellow pigment production by M. purpureus TISTR 3003 under SSF using ultrasound assisted pretreatment of SCB in the substrate preparation prior fermentation process. This research was performed according to central composite design and response surface methodology to identify the optimum key factors influencing the ultrasound assisted pretreatment factors such as H2O2 concentration, amplitude, SCB dosage and sonication time for better substrate preparation and subsequent fermentation process for enhancing the production of yellow pigments. The optimum conditions (2.5% H2O2, amplitude 79.8 µm, 3% SCB dosage and sonication time of 45 min) resulted in maximum yellow pigments production of 363.33 unit/g DW. The quadratic model equation of yellow pigments production showed high values of predicted R2 of 0.8109 and adjusted R2 of 0.9283. The interactions between the amplitude and sonication time were found to be insignificant in the production of yellow pigments. After validation of the model, based on the optimum conditions; H2O2 concentration, amplitude, SCB dosage and sonication time were found to be 2.74%, 83.22 µm, 2.84% and 52.29 min, the yellow pigments content was found 366.06±12.59 unit/g DW which is 2.59 times higher than untreated SCB. The experimental data suggest that ultrasound pretreatment is a valuable tool in the preparation of SCB as the substrate for enhanced yellow pigments production. Keyword: Monascus purpures, Pretreatment, Sugarcane bagasse, Ultrasound, Yellow pigment O-025 Enhancing the production of Monascus yellow pigments under solid state fermentation by ultrasound assisted pretreatment of sugarcane bagasse

173 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Khanita Kamwilaisak1*, and Wanwipa Kaewpradit2 1 Department of Chemical Engineering, Faculty of Engineering, Khon Kaen University, Khon Kaen, 40002, Thailand 2Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, *Correspondence to: Department of Chemical Engineering, Faculty of Engineering, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail:[email protected] O-031 The extraction of reducing sugars from post-harvest sugarcane waste by H2SO4 hydrolysis 1. INTRODUCTION Sugarcane (Saccharum officinarum) is amajorrawmaterialforsugar production inThailand, which is considered a large-scale industry in the country, accounting for 16% of the total industries in Thailand (Plangklang et al., 2012). The main product is either raw sugar or refined sugar, with a production capacity of up to 9.7 million tons per year, and there is a growing trend in sugar production (Office of the Cane and Sugar Board, Ministry of Agriculture and Cooperatives). However, when harvesting sugarcane, only a part of the plant can be utilized, specifically the stalks that contain sugar. Other parts, such as sugarcane leaves tops and shots become agricultural waste. Additionally, when sugarcane stalks are squeezed to extract sugarcane juice for sugar production, the remaining residue known as bagasse also becomes agricultural waste, and its quantity increases with the sugar production capacity. Finding solutions to manage and create value from agricultural waste generated by sugarcane cultivation (sugarcane leaves, bagasse, and tops) is crucial in helping Thai farmers utilize this waste. Moreover, it incentivizes farmers to reduce the burning of sugarcane leaves, which is an environmental issue. Hydrolysis is a chemical process involving a chemical compound’s reaction with water, where water molecules replace existing molecules within the compound, leading to decomposition or dissolution. Hydrolysis can be carried out using acids, bases, or water molecules themselves as the molecules that enter the reaction to break down the molecular composition of various chemical components of cellulose, hemicellulose, and lignin, which are the main components found in plant matter. This process commonly extracts sugar or water-soluble organic compounds from agricultural waste. For example, in a study by Jutakridsada et al. (2019), sugar was extracted from sugarcane leaves using hydrolysis with sulfuric acid. The resulting sugar content was 17.07 g/L, with glucose (C6) accounting for 2.59 g/L and xylose (C5) accounting for 14.48 g/L. Thus, this project aims to obtain the chemical and physical properties of sugarcane wastes including leaves, bagasse, primary, secondary, and tertiary shoots. Their properties are essential for designing conversion processes. Sulfuric hydrolysis was used to extract reducing sugar from sugarcane wastes. Thermogravimetric analysis, Bomb calorimeter and CHON analyses were performed on sugarcane wastes before and after hydrolyzing. 2. MATERIALS AND METHODS The agricultural waste from sugarcane, including sugarcane leaves, bagasse, and shoots, underwent several processing steps. Firstly, the waste was washed and dried. The dried sample wasthen blended and sieved using a mesh size of 20-80 (850 µm). The resulting sieved sample, with a concentration of 100g/L, was hydrolyzed using an aqueous solution of H2SO4 with a concentration ranging from 0 to 7%wt. The hydrolysis process took place at a temperature of 120°C for 60 minutes. After hydrolysis, the solid residue was separated through centrifugation at a speed of 5000 rpm for 15 minutes. The supernatant obtained from the centrifugation contained the extracted sugars, which were further analyzed for quantity and quality using HPLC (High-Performance Liquid Chromatography). Meanwhile, the solid residue was dried, and its heating value, chemical composition, and thermal gravimetric properties were determined. These analyses provide insights into the energy potential and composition of the remaining solid waste after hydrolysis.

174 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS 3. RESULTS AND DISCUSSION Figures 1 (a) to (e) illustrate the extraction of reducing sugars from sugarcane leaves, bagasse, and primary, secondary, and tertiary shoots using various H2SO4 aqueous solutions. It is evident that the extraction of reducing sugars gradually increased as the concentration of H2SO4 aqueoussolutionsincreased from 0 to 7%wt. The mechanism of sugar extraction through H2SO4 hydrolysis can be attributed to the hydrolyzing action of hydrogen ions from H2SO4, which break down and cleave the glycosidic bonds of lignin and hemicellulose molecules. This process leads to the release and dissolution of sugars or soluble compounds in the liquid phase. When the H2SO4 concentration is 0%wt, indicating the use of only distilled water, sugar groups and other compounds are extracted and dissolved back into the liquid. However, as the acid concentration increases, the hydrolyzing hydrogen ions from the acid effectively dissolve and extract sugar molecules, which can be subsequently recovered. It should be noted that increasing the acid concentration may also induce chemical reactions that convert sugar groups into other compounds, such as furfural, acetic acid, or levulinic acid (Chokesawatanakit et al., 2021). Consequently, this can result in a relatively lower measured sugar content despite the overall increase in reducing sugar extraction. The highest totalsugar content observed in sugarcane leaves was determined to be 21.47 ± 0.67 g/L, comprising 15.54 g/L of sucrose and 5.93 g/L of glucose. Figure 2 presentsthe thermal gravitational analysis(TGA) ofsugarcane leaves before and after H2SO4 hydrolysis. It can be divided into 4 stages; dehydration (50-245°C), degradation of residual sucrose (245 to 347 °C) (Cruz et al., 2013), thermal degradation of carbonaceous compounds, including lignin and possibly some hemicellulose (347 to 624°C) and ash (higher 624°C). The hydrolysis process does not affect the thermal degradation of sugarcane leaves because the TGA curve was similar before and after the hydrolysis reaction. Table 1 shows the elemental composition of sugarcane leaves and bagasse before and after H2SO4 hydrolysis. The elemental composition of biomass serves as fundamental data for selecting appropriate biomass conversion processesto maximize the benefits. The main elemental component ofsugarcane leaves and bagasse before hydrolysis with acid is carbon, ranging from 43.42% to 44.72%. Oxygen content is approximately similar, ranging from 43.76% to 44.51%. The remaining components are hydrogen, ranging from 5.52% to 5.93%, and nitrogen, with the lowest quantity ranging from 0.26% to 0.45%. After undergoing H2SO4 hydrolysis, the chemical composition of sugarcane leaves and bagasse shows an increased carbon content, ranging from 46.91% to 47.65%, with an increase of 2.59% to 3.49%. Meanwhile, the oxygen component decreases, ranging from 41.14% to 43.69%, with a reduction of 0.72% to 2.1%. The nitrogen component also decreases, ranging from 0.23% to 0.28%, with a reduction of 0.20% to 0.27%. The hydrogen component remains almost unchanged, with an increase of 0.01% to 0.23%. The increase in carbon content due to the hydrolysis process leads to an increase in the calorific value of sugarcane leaves and bagasse, a component, similar to the effect of torrefaction, a heat-based chemical process used to produce solid biofuels. Table 2 presents the calorific value of sugarcane leaves and bagasse before and after H2SO4 hydrolysis. It was found that the calorific values of the bagasse were similar in each growth cycle. Before hydrolysis, the calorific value ranged from 14.12 to 15.29 kJ/g, while after hydrolysis, it ranged from 15.38 to 15.96 kJ/g, slightly higher. The increased calorific value corresponds to the results of heat degradation tests, indicating that extracting sugar from the leaves and bagasse does not lead to a loss of calorific value but actually increases the calorific value due to the removal of volatile compounds. This process not only preserves the calorific value of the biomass but also allows for the conversion of the extracted substances into valuable chemicals for further use.

175 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Figure 1. Reducing sugars from (a) sugarcane leaves, (b) bagasse, (c) primary shoots, (d) secondary shoots, and (e) thirdly shoots using various H2SO4 aqueous solutions. Figure 2. Thermal gravitational analysis of sugarcane leaves before and after H2SO4 hydrolysis

176 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Table 1. Element composition of sugarcane leaves and bagasse before and after H2SO4 hydrolysis Table 2. The calorific value of sugarcane leaves and bagasse before and after H2SO4 hydrolysis. 4. CONCLUSION Agricultural waste from sugarcane can be raw material to extract the reducing sugar by H2SO4 hydrolysis. The highest total sugar content observed in sugarcane leaves was determined to be 21.47 ± 0.67 g/L, comprising 15.54 g/L of sucrose and 5.93 g/L of glucose. Furthermore. this research demonstrates the effect of the calorific value of waste bagasse from sugarcane before and after the acid hydrolysis process, and it reveals an increase in the calorific value. Therefore, it is necessary to study the potential of using waste bagasse from sugarcane as a high-quality fuel pellet that can be compressed into solid fuel. This is because volatile compounds are eliminated during the acid hydrolysis process, thereby enhancing the combustible potential of the biomass. 5. ACKNOWLEDGMENT The authors would like to express appreciation for the support of the sponsors by the National Research Council of Thailand (2021). The authors would also like to thanks to the Northeast Thailand Cane and Sugar Research Center (NECS), Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand. 6. REFERENCES Chokesawatanakit, N., Jutakridsada, P., & Kamwilaisak, K. (2021). Sustainable valorization of sugarcane leaves for succinic acid and biochar production. Journal of Metals, Materials and Minerals, 31(2), 46-53. https://doi. org/10.55713/jmmm.v31i2.1048 Cruz, G., Monteiro, P. A., Braz, C., Seleghim Jr, P., Polikarpov, I., & Crnkovic, P. M. (2013). Investigation of porosity, wettability and morphology of the chemically pretreated sugarcane bagasse. 22nd. International Congress of Mechanical Engineering. Ribeirão Preto, SP, Brazil, Jutakridsada, P., Saengprachatanarug, K., Kasemsiri, P., Hiziroglu, S., Kamwilaisak, K., & Chindaprasirt, P. (2019). Bioconversion of Saccharum officinarum Leaves for Ethanol Production Using Separate Hydrolysis and Fermentation Processes. Waste and Biomass Valorization, 10(4), 817-825. https://doi.org/10.1007/ s12649-017-0104-x Plangklang, P., Reungsang, A., & Pattra, S. (2012). Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse. International Journal of Hydrogen Energy, 37(20), 15525-15532.

177 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Ratchanok Khongchum¹, Chotiwit Sriwong¹, and Prakit Sukyai2 * 1 Cellulose for Future Materials and Technologies Special Research Unit, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 50 Ngamwongwan Road Chatuchak, Bangkok, 10900, Thailand ²Center for Advanced Studies for Agriculture and Food (CASAF), Kasetsart University Institute for Advanced Studies, Kasetsart University, 50 Ngamwongwan Road, Chatuchak, Bangkok, 10900, Thailand *Correspondence to: Cellulose for Future Materials and Technologies Special Research Unit, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 50 Ngamwongwan Road Chatuchak, Bangkok, 10900, Thailand. [email protected] ABSTRACT: In thisstudy,sugarcane bagasse (SCB)was used as a rawmaterialto extract cellulose, cellulose is one of the most well-known and widely used materials in bone tissue engineering. However, there are still certain limitationsto the cellulose-based cellularstructure. Especially promote MC3T3-E1 osteoblasts. This study aimsto study the effect of addingmaterialsthat can promote cell growth. Hydroxyapatite and silkfibroin were added to regenerated cellulose extracted from sugarcane bagasse to promote and induce bone formation of osteoblasts and compare the difference between regenerated cellulose with hydroxyapatite scaffold and regenerated cellulose with hydroxyapatite and silk fibroin at different concentration. Regenerated cellulose prepared from cellulose, cellulose extract from sugarcane bagasse by steam explosion and bleaching with sodium chlorite. Then dissolve pure cellulose fibers with 1-butyl-3-methylimidazolium chloride, followed by mixing of hydroxyapatite into the cellulose solution. Regenerated cellulose/hydroxyapatite hydrogel was immersed in a silk fibroin solution extracted from Bombyx mori mixed with hydroxyapatite. at different concentrations to coat regenerated cellulose/hydroxyapatite hydrogel and freeze-dry to obtain scaffolds. The results show that scanning electron microscopy shows the porous structure of the scaffold, appropriate for cellular activities. The Fourier transform infrared spectroscopy, Energy-dispersive X-ray spectrometry and X-ray diffraction analysis confirm of cellulose hydroxyapatite and silk fibroin composition. In addition, after culturing MC3T3-E1 osteoblasts cells on the scaffolds, Scaffold can support cell proliferation and non-toxic to cells. That indicates, hydroxyapatite and silk fibroin enhance cells proliferation and adhesion ability. Therefore, this research demonstrates the properties of the synthesized scaffold that can be applied in tissue engineering. Keyword: Scaffold, Regenerated cellulose, Hydroxyapatite, Silk fibroin, Bone tissue engineering. O-038 Value addition of sugarcane bagasse in scaffold production of regenerated cellulose incorporated with hydroxyapatite and silk fibroin.

178 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Ekabhop Arunmas¹, Akkaratch Rodklongtan¹ and Pakamon Chitpresert1 * 1 Technological Innovation of probiotics and plant extracts for functional food special research unit, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand *Correspondence to: Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok,10900, Thailand. E-mail: [email protected] ABSTRACT: Sugarcane bagasse, a major byproduct from the sugar industry, is mainly used to generate combustion fuel, but its economic value has not been maximized. This research added value to sugarcane bagasse by optimizing vitamin D-loaded lignin nanoparticle formation using antisolvent nanoprecipitation combined with ultrasonication. The process parameters comprised the order of solvent-antisolvent addition, ultrasonication amplitude and lignin type i.e., alkaline lignin (extracted from sugarcane bagasse), indulinAT and sodium lignosulfonate. Results showed that the optimal condition was the addition of solvent (alkaline lignin solution) to the anti-solvent (deionized water) at 30% amplitude yielding vitamin D-loaded lignin nanoparticles with suitable size, polydispersity index and zeta-potential for vitamin D delivery (115 nm, 0.126 and -10.6 mV, respectively). Highest vitamin D encapsulation efficiency and loading capacity were 91% and 16%, respectively. Optimized vitamin D-loaded lignin nanoparticles exhibited pH-dependent and controlled release behavior during in vitro gastrointestinal digestion. During passage through a dynamic in vitro gastrointestinal model, only 14% of vitamin D was released in the simulated gastric fluid, with the remaining amountsubsequently released in simulated intestinal fluid. Vitamin D-loaded lignin nanoparticles formed using the commercial lignins indulin AT and sodium lignosulfonate had larger particle size and higher polydispersity index and were not suitable for drug delivery, with significantly lower (p < 0.05) encapsulation efficiency and loading capacity. Lignin functional groups were elucidated by 31P-NMR. Result showed the high potential of alkaline lignin from sugarcane bagasse for the development of vitamin D nanoparticle delivery systems. Keyword: Sugarcane bagasse, Lignin, Lignin nanoparticles, Vitamin D, Drug delivery. O-040 Development of lignin nanoparticles from sugarcane bagasse for vitamin D delivery

179 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS 1. INTRODUCTION Sugarcane (Saccharum officinarum) is the main raw material for sugar production. Typically, 30-34 tons of sugarcane bagasse are generated from processing 100 tons of sugarcane. This byproduct is usually burned as low grade fuel for energy recovery. Lignin is a natural phenolic polymer and one of the main components in sugarcane bagasse. Lignin consists of phenylpropane units linked by three different monolignols as p-hydroxyphenyl, guaiacyl and syringyl moieties. These natural phenolic compounds exhibit potential bioactivity [1]. Lignin nanoparticles (LNPs) exist as nanosized structured biopolymer aggregations formed by the complicity of the hydrophilic and hydrophobic lignin functional groups. The lignin hydrophobic functional groups are positioned inside the formed aggregated structure and covered by the phenolic and aliphatic hydroxyl lignin hydrophilic functional groups. Thus, LNPs, as an amphiphilic polymer are a good choice as a carrier of a hydrophobic molecule that can be exposed in high-polarity environments. LNPs also have excellent properties, such as renewability, sustainability, biodegradability, biocompatibility, and safety [2]. Vitamin D is an essential vitamin that plays a beneficial role in human bone and teeth development. New evidence has suggested a relationship between vitamin D consumption and reduction of chronic diseases such as autoimmune disorders, diabetes mellitus, cardiovascular diseases, cancers, and inflammatory diseases. However, vitamin D is poorly soluble in water, making it difficult to incorporate in food formulations. Crystalline forms of vitamin D also have low bioavailability when administered orally, with low solubility in the human gut. Vitamin D deficiency has now become a global issue [3]. This research optimized the process of vitamin D-loaded lignin nanoparticle formation using antisolvent nanoprecipitation combined with ultrasonication. The process parameters comprised the order of solvent-antisolvent addition, ultrasonication amplitude and lignin type i.e., alkaline lignin (extracted from sugarcane bagasse), indulin AT (USA) and sodium lignosulfonate (China). Vitamin D was chosen as hydrophobic drug for encapsulation by lignin. After successful drug encapsulation, particle size, polydispersity index, zeta-potential, and in vitro release properties of vitamin D-loaded lignin nanoparticles were determined. The impact of functional lignin groups on the performance of LNPs was also analyzed by 31P nuclear magnetic resonance (31P NMR). 2. MATERIALS AND METHODS Materials Sugarcane bagasse was provided by Kaset Thai International Sugar Corporation Public Co., Ltd. (Nakhon Sawan, Thailand). The bagasse was thoroughly washed with water, and then dried in a hot air oven at 60 °C. The dried sample was milled to an average size of 0.125 mm and stored at -20 °C until further use. Indulin AT was kindly supplied by Ingevity (North Charleston, S.C., USA) and sodium lignosulfonate was sourced from Binzhou Chengli Building Materials (China). Sodium xylenesulfonate (SXS) was purchased from JKK Chemical (Bangkok, Thailand). Alkaline lignin extraction Alkaline lignin was obtained by alkaline extraction from sugarcane bagasse at 95 °C with 1% sodium hydroxide (w/v) for 1 h and 1:20 (w/v) liquid: solid ratio, in a 2 L stirred reactor. The lignin in the black liquor was precipitated with 50% sulfuric acid until reaching pH 2. The lignin was centrifuged, washed with distilled water to reach neutral pH, and dried in an oven at 60 °C. Vitamin D-loaded lignin nanoparticle formation Vitamin D-loaded lignin nanoparticles were synthesized using the antisolvent nanoprecipitation method. Lignin (5 mg/ml) was dissolved in 1.92 M sodium xylenesulfonate (SXS) under overhead stirring (IKA, Germany) for 30 min. Vitamin D (6 mg) was dissolved in 95% ethanol and added to the lignin-SXS solution with overhead stirring (IKA, Germany) for 30 min. Subsequently, lignin-vitamin D in SXS was diluted with deionized water to SXS concentration of 0.3 M. The suspended vitamin D-loaded lignin nanoparticles were separated using centrifugation (Sorvall, Thermo Scientific, USA) and washed two times with deionized water to remove SXS. The centrifuged vitamin D-loaded lignin nanoparticles were then redispersed with deionized water, ultrasonicated and freeze dried. Nanoparticle formation optimization was achieved by changing the formation parameters, i.e., order ofsolvent-antisolvent addition, ultrasonication amplitude and lignin type. Characterization Particle size, polydispersity index and zeta-potential were characterized using a Zeta sizer Nano ZS (Malvern Instruments, UK), with functional lignin groups analyzed by using 31P NMR (Bruker Avance III HD 500 MHz). 31P

180 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS NMR samples were prepared and analyzed following the procedure reported byArgyropolous et al. [4]. Encapsulation efficiency, loading capacity and solubilized vitamin D concentration were evaluated using High-Performance Liquid Chromatography (HPLC) (Shimadzu Model LC-20AT, Tokyo, Japan) as described by Melo et al. [5]. Calculations were conducted according to equation 1-3. In-vitro gastro-intestinal tract release was performed according to Lin et al. [6]. where, Wd = weight of vitamin D loaded in lignin nanoparticles. Wdi = weight of vitamin D added initially. Ws = weight of total solid content. V = total volume 3. RESULTS AND DISCUSSION Effects of process parameters Results of the order of solvent-antisolvent addition are shown in Table 1. In method 1, lignin-vitamin D solution as solvent; S was added to deionized water as antisolvent; A (S → A). In method 2, the addition in opposite order or vice versa as A → S was performed. Smaller particle size and narrower polydispersity index of vitamin D-loaded lignin nanoparticles were obtained from S → A. The change in particle size was explained as follows: for S → A, a relatively high value of antisolvent was achieved in a localized volume at the point of introduction of the solution as soon as it came in contact with water that was present in excess. Dilution of the lignin-vitamin D solution by the antisolvent occurred promptly and fastsupersaturation took place. The high rate of nucleation resulted in the formation of a large number of nuclei, thereby producing smaller particles. By contrast, for A → S, when the antisolvent was continuously injected, the lignin-vitamin D solution became cloudy after an adequate amount of antisolvent was added. This delayed solution nucleation, which in turn produced fewer nuclei, generating larger individual particles. Zeta-potential values obtained for S → A and A → S were -10.6 ± 0.46 and -2.99 ± 0.45 mV, respectively. Slightly negatively charged particles reduce the undesirable clearance by the reticuloendothelial system (RES) and improve the blood compatibility through longer circulation in the blood stream [7]. S → A had a significantly higher encapsulation efficiency than A → S, which was explained by the same theory as described above. S → A had a high supersaturation rate and nuclei formation resulting in better π–π interactions and hydrophobic interactions between lignin and vitamin D. The ultrasonication amplitude had no effect on particle size and zeta-potential. By contrast, the polydispersity index increased (Table 1) because at higher amplitude, the cavitation rate of formation, growth, and collapse of bubbles also increased. Cavitation causes microjets and shock waves [8] thit can result in extensive oxidation, producing radicals that initiate radical-induced repolymerization. Phenolic hydroxyl (OH) groups in lignin form phenoxy radicals during sonication and may induce cross-linking reactions causing lignin nanoparticles to agglomerate [9]. The encapsulation efficiency of vitamin D-loaded lignin nanoparticles progressively increased at higher amplitude, reaching the maximum (89.42%) at 30% amplitude. When the amplitude was further increased to 40%, the encapsulation efficiency decreased significantly, indicating that excessive amplitude was not conducive to intermolecular interaction between lignin and vitamin D from repolymerization as mentioned above. VitaminD-loaded lignin nanoparticlesformedusing commercialligninsi.e.,indulinATandsodiumlignosulfonate had larger particle size (≥ 150 nm) and a higher polydispersity index (≥ 0.3) than alkaline lignin and were not suitable for drug delivery, while encapsulation efficiency and loading capacity were significantly lower (Table 1). Indulin AT and sodium lignosulfonate have high polar functional groups such as carboxylic, phenolic and aliphatic hydroxyl groups. As a result, the hydrophilic structure of lignin could not be precipitated by deionized water and produced large particles. This phenomenon can be explained by two theories: 1) a hydrophilic lignin structure causes slow nucleation and supersaturation rates and, 2) carboxylic, phenolic and aliphatic hydroxyl groups cannot self-assemble and do not form as lignin nanoparticles [10].

181 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Table 1 Effects of the procedure parameters on particle size, polydispersity index (PDI), zeta-potential and *Different letters in the same parameter and column indicate significant difference (p < 0.05). Indulin AT and sodium lignosulfonate had lower encapsulation efficiency, loading capacity, solubilized vitamin D concentration and yield than alkaline lignin, as shown in Figure 1. Because alkaline lignin is more hydrophobic, vitamin D can be encapsulated more efficiently than indulinAT and sodium lignosulfonate.According to the extremely low water solubility of vitamin D, this application was limited; however, the solubility of encapsulated vitamin D improved. When compared to the free vitamin D concentration in water (0.5 µg/ml), the solubilized vitamin D concentration in vitamin D-loaded alkaline lignin nanoparticles showed a 179-fold increase. The hydrophobicity and hydrophilicity of lignin were the main factors that influenced lignin nanoparticle properties. The functional groups of lignin are elucidated in the next section. Figure 1 Effects of lignin types on yield, encapsulation efficiency (EE), loading capacity (LC) and solubilized vitamin D concentration of vitamin D-loaded lignin nanoparticles formed using alkaline lignin (VitD-AL-NPs), indulin AT (VitD-AT-NPs) and sodium lignosulfonate (VitD-SL-NPs) *Different letters in the same response indicate significant difference (p < 0.05).

182 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Functional groups The phenolic hydroxyl, aliphatic hydroxyl, and carboxyl groups of the lignin samples were assessed by 31P NMR, with analysis results of alkaline lignin, indulin AT and sodium lignosulfonate summarized in Table 2. In lignin, higher hydroxyl content, notably phenolic hydroxyls, corresponds to higher hydrophilicity of the lignin molecule. Alkaline lignin had the lowest content of phenolic hydroxyl groups, syringyl guaiacyl and p-hydroxyphenyl, indicating its low polarity and hydrophobic structure. Indulin AT and sodium lignosulfonate contained higher amounts of phenolic hydroxyl groups, syringyl, guaiacyl and p-hydroxyphenyl, indicating a more polar and hydrophilic structure [11]. The hydrophobic and hydrophilic properties of lignin directly affect the properties of vitamin D-loaded lignin nanoparticles as explained in the above section. Table 2 Functional groups of alkaline lignin, indulin AT and sodium lignosulfonate analyzed by 31P NMR In vitro release The release profiles of vitamin D from lignin nanoparticles are shown in Figure 2a. For alkaline lignin, in simulated gastric fluid, the release rate of vitamin D was slow, with only 14.10% of vitamin D released after 2 h, indicating that vitamin D was well protected from the harsh stomach environment. In simulated intestinal fluid, 86% of vitamin D was released after 1 h and constantly released for 4 h. The release rate of vitamin D was greatly accelerated under the simulated intestinal condition. Indulin AT also protected vitamin D in the stomach, with only 9.01% released after 2 h while sodium lignosulfonate released vitamin D in simulated gastric and intestinal fluid at 44.93% and 78.19%, respectively. Releasing high vitamin D in the stomach caused loss of vitamin D through deterioration. Alkaline lignin and indulin AT were able to store vitamin D in the simulated stomach; but alkaline lignin significantly released vitamin D in the small intestine where it was absorbed. The higher ionizable carboxyl groups in alkaline lignin (Table 1) broke down the lignin structure in the intestine [12]. To consider environmental changes, the in vitro drug release behavior of vitamin D-loaded lignin nanoparticles was investigated in simulated gastric fluid for 2 h, after which they were transferred to the simulated intestinal fluid. Results of this “biomimetic” evaluation indicated that the drug release profile of vitamin D-loaded alkaline lignin nanoparticles was pH-dependent, as described above. Release rate in the simulated intestinal fluid was higher than in simulated gastric fluid, enabling drug delivery or release to occur preferentially in the intestine while avoiding drug leakage in the stomach. Thus, the release profiles of vitamin D from alkaline lignin met the release requirements of oral administration. Furthermore, results in Figure 2b confirmed that released vitamin D in the small intestine was not degraded and could be absorbed through the intestinal membrane via clathrin and caveolin mediated endocytosis.

183 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Figure 2 Simulated gastro-intestinal (a) release of vitamin D-loaded lignin nanoparticles formed using alkaline lignin (VitD-AL-NPs), indulin AT (VitD-AT-NPs) and sodium lignosulfonate (VitD-SL-NPs) and (b) stability of free vitamin D 4. CONCLUSIONS Vitamin D-loaded lignin nanoparticles were successfully developed for efficient drug delivery. Vitamin D-loaded lignin nanoparticlesformed with alkaline lignin,solvent to anti-solvent (S →A) and 30% amplitude exhibited suitable particle sizes (≤ 150 nm) and zeta-potential (± 10 mV), essential for absorption through the intestinal membrane and evading the reticuloendothelial system. The in vitro release behavior of vitamin D from lignin nanoparticles exhibited pH dependence. Less than 14% of the loaded vitamin D was released in the simulated gastric fluid, while nearly all of the initial drug content was released in the simulated intestinal fluid. This system showed good prospects for using lignin from sugarcane bagasse in the controlled delivery of other hydrophobic oral drugs such as curcumin and resveratrol. 5. ACKNOWLEDGEMENT This work was financially supported by the National Research Council of Thailand. 6. REFERENCES [1] Qin Z., Liu H. M., Gu L. B., Sun R. C. and Wang, X. D. (2020). Lignin as a Natural Antioxidant: Property-Structure Relationship and Potential Applications. In Gutiérrez T.J. (eds) Reactive and Functional Polymers Volume One, Springer, Cham. https://doi.org/10.1007/978-3-030-43403-8_5 [2] Wijaya C.J., Ismadji S. and Gunawan S. (2021). A Review of Lignocellulosic-Derived Nanoparticles for Drug Delivery Applications: Lignin Nanoparticles, Xylan Nanoparticles, and Cellulose Nanocrystals. Molecules. 26, 676. https://doi.org/10.3390/molecules26030676 [3] Walia N. and Chen L. Pea protein based vitamin D nanoemulsions: Fabrication, stability and in vitro study using Caco-2 cells. Food Chemistry. 305 (125475). https://doi.org/10.1016/j.foodchem.2019.125475 [4] Argyropoulos D.S., Pajer N. and Crestini C. (2021). Quantitative 31P NMR Analysis of Lignins and Tannins. J. Vis.Exp. (174), e62696, doi:10.3791/62696. [5] Melo A. P. Z., Rosa C. G., Noronha C. M., Machado M. H., Sganzerla W. G., Bellinati N. V. and Barreto P. L. M. (2021). Nanoencapsulation of vitamin D3 and fortification in an experimental jelly model of Acca sellowiana: Bioaccessibility in a simulated gastrointestinal system. LWT, 145(3), 111287. https://doi. org/10.1016/j.lwt.2021.111287. [6] Lin X., Chen P. X., Robinson L. E., Rogers M. A., and Wright A. J. (2021). Lipid digestibility and bioaccessibility of a high dairy fat meal is altered when consumed with whole apples: Investigations using static and dynamic in vitro digestion models. Food Structure, 28. https://doi.org/10.1016/j.foostr.2021.100191. [7] Sadat S.M.A., Jahan S.T. and Haddadi, A. (2016). Effects of Size and Surface Charge of Polymeric Nanoparticles on in Vitro and in Vivo Applications. Journal of Biomaterials and Nanobiotechnology, 7, 91-108. http://dx.doi.org/10.4236/jbnb.2016.72011 [8] Ni Y., Li J., Fan L. (2021). Effects of ultrasonic conditions on the interfacial property and emulsifying property of cellulose nanoparticles from ginkgo seed shells. Ultrasonics – Sonochemistry. 70 (105335). https://doi. org/10.1016/j.ultsonch.2020.105335

184 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS [9] Agustin M.B., Penttilä P.A., Lahtinen M., and Mikkonen K.S. (2019). Rapid and Direct Preparation of Lignin Nanoparticles from Alkaline Pulping Liquor by Mild Ultrasonication. ACS Sustainable Chemistry & Engineering 7 (24), 19925-19934 DOI: 10.1021/acssuschemeng.9b05445 [10] Zwilling J.D., Jiang X., Zambrano F., Venditti R.A., Jameel H., Velev O.D., Rojas O. J., Gonzalez R. (2021). Understanding lignin micro-and nanoparticle nucleation and growth in aqueous suspensions by solvent fractionation. Green Chem. 23 (2), 1001–1012. https://doi.org/10.1039/D0GC03632C [11] Yang Y., Xu J., Zhou J. and Wang X. (2022). Preparation, characterization and formation mechanism of size-controlled lignin nanoparticles. International Journal of Biological Macromolecules 217. https://doi. org/10.1016/j.ijbiomac.2022.07.046 [12] Li Y., Qiu X., Qian Y., Xiong W. and Yang D. (2017). pH-responsive lignin-based complex micelles: preparation, characterization and application in oral drug delivery, Chemical Engineering Journal (327). doi: http://dx.doi.org/10.1016/j.cej.2017.07.022

185 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Parawee Pumwongpitak1 *, Saengdoen Daungdaw¹, Laksana Wangmooklang¹, Busarin Noikeaw¹, Thammasit Vongsetkul² and Siriporn Larpkiattaworn¹ 1 Expert Centre of Innovative Materials, Thailand Institute of Scientific and Technological Research, Pathum Thani, 12120, Thailand ²Faculty of Science, Mahidol University, Bangkok, 10400, Thailand *Correspondence to: Expert Centre of Innovative Materials, Thailand Institute of Scientific and Technological Research, 35 Mu. 3, Pathum Thani, 12120, Thailand. [email protected] ABSTRACT: Sugarcane bagasse, the abundant waste material from sugarcane milling, is a carbon resource from biomass. In this study, porous carbon material derived from sugarcane bagasse was produced using the carbonization process, followed by the alkali chemical activation process. The bagasse was carbonized at 400°C for 1 h under a nitrogen atmosphere. After that, the activation process was performed using KOH solution as an activating agent at various concentrations to create a porous structure resulting in a high surface area. The activation temperatures at 600, 700, and 800°C for 2 h were investigated under a nitrogen atmosphere. The yield of obtained carbon from carbonization and activation processes was calculated. The as-prepared porous carbon was characterized by Brunauer-Emmet-Teller (BET) to determine the carbon's surface area and pore size. The scanning electron microscope (SEM) evaluated the morphology and microstructure of the obtained porous carbon. Keyword: Sugarcane bagasse, Carbonization, Activation, Porous carbon, Surface area P-010 Preparation and characterization of Porous Carbon from Sugarcane Bagasse

186 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS 1. INTRODUCTION Sugarcane bagasse is the fibrous residue that remains after sugarcane juice has been extracted in the sugar mill process. A ton of sugarcane can generate about 280 kg of bagasse [1]. The sugarcane bagasse is composed of 26-47% of cellulose, 19-33% of hemicellulose, 14-2% of lignin, and 1-5% of ash [2]. The Sugarcane bagasse is a carbon-rich biomass that is abundant, environmentally friendly, inexpensive, and sustainable. Therefore, it is suitable for producing porous carbon material [3,4]. In general, the activated or porous carbon can be synthesized using physical or chemical activation. However, the chemical activation process leads to a higher surface area and requires a shorter time than the physical method. The chemicals that can be used for impregnation for the chemical activation process can be acids, alkalis, or oxidants [3]. In this work,sugarcane bagasse from the sugar mill industry is used asraw material for preparing bagasse carbon. The samples are carbonized under a nitrogen atmosphere and then treated with Potassium hydroxide solution before activation at various temperatures. The surface area and morphology properties of treated samples are investigated using BET analysis and scanning electron microscope (SEM). 2. MATERIALS AND METHODS 2.1 Materials In this experiment, the sugarcane bagasse samples were obtained from the sugar refinery of Saraburi Sugar Co., Ltd, Saraburi, Thailand. The alkali that was used in the chemical activation process was potassium hydroxide (KOH) from KEMAUS, Elago Enterprises Pty Ltd, Cherrybrook, New South Wales, Australia. The hydrochloric acid was purchased from RCI Labscan Limited, V.S. Chem House, Krung Thep Maha Nakhon, Thailand. 2.2 Methods The raw sugarcane bagasse was ground and washed with distilled water. Then, the dried samples were carbonized in the tube furnace at 400 °C for 1 hour with a ramp rate of 5 °C per minute under a nitrogen atmosphere (SBC). After that, the carbon activating process was conducted using the chemical activation process. The carbonized samples were immersed in 3 M and 5 M KOH solutions for 16 hours (SBC3M and SBC5M). Then, the samples were heated in the tube furnace at 600, 700, and 800 °C for 2 hours with a ramp rate of 5 °C per minute under a nitrogen atmosphere. The activated samples were neutralized using diluted hydrochloric acid and then washed with distilled water until their pH was neutral. The products were dried in an oven at 80 °C for 12 hours. The samples were characterized using Autosorb IQ, Quantachrome Instruments for Brunauer-Emmet-Teller (BET) surface area, pore size, and pore volume. The morphology of samples was investigated using SEM (JSM-5410LV, JEOL). 3. RESULTS AND DISCUSSION The average yield of the samples in the carbonization process at 400 °C was 7.89 %, while the weight loss was an average of 92.11%. The lost mass during the carbonization process occurred because of cellulose and lignin decomposition in bagasse. In the activating process, the weight loss of activated samples treated at 700 °C and by 3 M and 5M KOH were 14.21 % and 24.51 %, respectively. It indicates that weight loss increases with increasing the KOH concentration due to the pore formation of carbon. 3.1 The surface area of the samples The surface area of the carbonized bagasse sample from the refining factory was 0.34 m2/g. When the bagasse samples were activated using KOH solution, the surface area values of treated samples increased higher than the carbonized sample, as seen in Table 1. The surface area of the carbonized sample treated with 3 M KOH and heated at 600 °C was 10.76 m2/g. As the concentration of the KOH solution increased from 3 M to 5 M, a surface area of 43.11 m2/g can be obtained. Moreover, the sample treatment with 5 M KOH and heat at 700 °C) significantly improved the surface area value to 150.04 m2/g because the new micropores and mesopores were generated [4]. However, immersing the sample in 3 M KOH and then activating it at 700 °C resulted in no difference between the surface area values of the sample compared to 600 °C. Table 1 Surface area values of activated samples at various conditions

187 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Table 2 demonstratesthe average pore diameter of the activated samples. It can be evidenced that the pore volume of SBC3M and SBC5M treated at 600 °C was increased from 0.03 cm3/g to 0.07 cm3/g when the concentration of KOH solutions increased from 3 M to 5 M. Regarding activation temperature, the pore volume of SBC5M samples treated at 600 °C and 700 °C also increased from 0.07 cm3/g to 0.13 cm3/g. In contrast, the average pore diameter decreased with the increasing concentration of KOH and activated temperatures. The average pore diameters of SBC3M and SBC5M are 9.44 nm and 7.21 nm, which decrease with increasing KOH solution concentrations. The average pore diameter of SBC5M was also reduced from 7.21 nm to 4.78 nm when the activated temperature increased from 600 °C to 700 °C. Thus, the concentration of the KOH solution and the activated temperatures play essential roles in order to form the structure of the activated samples. Gu et al. studied the microporous bamboo char with the BET surface area of the carbonized and activated samples were 56.00 and 791.80 m2/g and the pore volume 0.05 and 0.38 cm3/g [5]. Table 2 Pore volume and average pore diameter values of activated samples at various conditions 3.2 Morphology The microstructure of the untreated sugarcane bagasse, carbonized, and activated samples were studied. Figure 2a shows untreated sugarcane bagasse with a smooth surface. When the bagasse was ground to a smaller size and carbonized, the rough surface on some of the particles can be observed, as we can see in Figure 2b. The activated samples (SBC3M at 600 °C, SBC5M at 600 °C, SBC3M at 700°C, and SBC5M at 700°C in Figures 2c, 2d, 2e, and 2f) show the morphology of irregular shapes with porous structures on the surfaces. However, the SBC3M at 800°C in Figures 2g and 2h illustrates the irregularly shaped edges and corners with smooth surfaces due to the pore collapse or destruction of the porosity. It suggests the sample's surface area could be lower than porous samples. Figure 1 SEM micrographs of (a) untreated sugarcane bagasse, (b) SBC, (c) SBC3M at 600 °C, (d) SBC5M at 600 °C, (e) SBC3M at 700°C, (f) SBC5M at 700°C, and (g and h) SBC3M at 800°C 4. CONCLUSIONS The activated carbon from sugarcane bagasse in a refining factory can be produced using carbonization and activation processes with KOH solution. The surface area of activated samples increases with increasing the KOH concentration and activation temperatures. The treated sample with 5 M KOH, then activated at 700°C, provides

188 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS a high surface area of 150.04 m2/g with a pore volume of 0.13 cm3/g and an average pore diameter of 4.78 nm. Therefore, sugarcane bagasse can be an alternative material for producing activated carbon. 5. ACKNOWLEDGEMENT We are grateful for the financial support from the National Research Council of Thailand (NRCT) and the Thailand Institute of Scientific and Technological Research (TISTR). We thank Saraburi Sugar Co., Ltd., in Saraburi, Thailand, for providing us with the raw material. 6. REFERENCES [1] Mkhwanazi, Z., Isa, Y. M., & Vallabh, S. T. (2023). Production of Biocoal from Wastewater Sludge and Sugarcane Bagasse: A Review. Atmosphere, 14(1), pp. 184. https://doi.org/10.3390/atmos14010184 [2] Mahmud, M. A., & Anannya, F. R. (2021). Sugarcane bagasse-A source of cellulosic fiber for diverse applications. Heliyon, 7(8), e07771. [3] Kakom, S. M., Abdelmonem, N. M., Ismail, I. M., & Refaat, A. A. (2023). Activated Carbon from Sugarcane Bagasse Pyrolysis for Heavy Metals Adsorption. Sugar Tech, 25(3), 619-629. doi:10.1007/ s12355-022-01214-3 [4] Raupp, Í. N., Valério Filho, A., Arim, A. L., Muniz, A. R. C., & da Rosa, G. S. (2021). Development and characterization of activated carbon from olive pomace: experimental design, kinetic and equilibrium studies in nimesulide adsorption. Materials, 14(22), 6820. [5] Gu, X., Wang, Y., Lai, C., Qiu, J., Li, S., Hou, Y., . . . Zhang, S. (2015). Microporous bamboo biochar for lithium-sulfur batteries. Nano Research, 8(1), 129-139. doi:10.1007/s12274-014-0601-1

189 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Anawin Junsawang1, Suphannika Intanon2,3 and Nichakorn Kondee1,3* 1Department of Natural Resource and Environment, Faculty of Agriculture, Natural Resource and Environment, Naresuan University, Phitsanulok, 65000, Thailand 2Department of Agricultural Science, Faculty of Agriculture, Natural Resource and Environment, Naresuan University, Phitsanulok, 65000, Thailand 3National Biological Control Research Center, Lower Northern Regional Center, Naresuan University, Phitsanulok, 65000, Thailand *Correspondence to: Department of Natural Resource and Environment, Faculty of Agriculture, Natural Resource and Environment, 99 Moo 9 Naresuan University, Phitsanulok, 65000, Thailand, E-mail : [email protected] ABSTRACT: Currently, sugarcane plantations are facing a decrease in production and quality due to the presence of weeds within the planting area. Weeds compete with the sugarcane for essential nutrients and water and serve as habitats for plant pests and diseases, leading to poorer quality sugarcane. This study aims to develop an effective biological weed control agent using a combination of natural substances, including a biosurfactant, lignin, sodium chloride, xanthan gum, vegetable oil and allelopathic substances extracted from mango or sugarcane leaves. The mixture of these substances was tested for stability for over a month and then applied to two-leaf seedlings of Bidens pilosa L., a common weed in sugarcane plantations. The mixture was sprayed on the weeds for three days, once a day, and observed for 14 days after spraying. It was found that the emulsion combined with the allelopathic substance extracted from mango or sugarcane leaves effectively inhibited weed growth. The mixture of the natural substances successfully inhibited the growth of B. pilosa by 100%, with the effects lasting for 14 days. This agent was then tested on weeds in an actual sugarcane crop to evaluate its efficacy in real situation. The results showed a tendency of effective inhibition against narrow-leaf and broad-leaf weeds. Moreover, it is safe for users and does not cause harm to the environment. Therefore, the mixture is a promising alternative to chemical herbicides for weed control in sugarcane plantations. Keyword: Bioherbicide Biosurfactant Invasive weed control Allelopathic Natural substances P-014 Development of a bioherbicide combining allelopathy extract form plant leaves with biosurfactant from Brevibacterium casei NK8

190 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS 1. INTRODUCTION Weed infestationsin the fields are the cause ofthe current decline in agricultural output.There may be competition for water and nutrients and covering of the primary crop. Therefore, farmers have to apply various ways to eliminate weeds from their crops. The majority of weed control techniques involve the use of various chemicals, which can be harmful to both the user and the environment [3]. Consequently, farmers have been focusing on safer herbicides. The usage of bioherbicides should be an important step towards sustainability in agriculture. Thisresearch aimsto develop a bioherbicide with highersafety for users, living organisms, and the environment. The bioherbicide will be formulated using safe and environmentally friendly ingredients, consisting of six components: 1) biosurfactant that reduces surface tension, enhances penetration into weed leaves; 2) lignin that acts as a co-surfactant and improving the efficiency of micelle and emulsion formation; 3) sodium chloride (NaCl) that reduces cell size, surface tension and enhances penetration into weed leaves; 4) vegetable oil that increases adhesion to plant leaves; 5) xanthan gum that helps stabilize emulsions and enhances persistence; and 6) plant leaf extracts (mango and sugarcane leaves) that contains active compounds for weed control [1] The developed bioherbicide was expected to be safe and non-toxic to both environment and farmer. It was developed to be as an alternative in weed control as chemical herbicides. All the ingredients will be formulated as an emulsion, ensuring compatibility and stability. This emulsification process allowed for better coverage of the weed leaves, reducing runoff after application, improving adhesion to weed leaves, and facilitating penetration into the weed tissue. These factors contributed to the bioherbicide's efficacy in controlling weed growth. 2. MATERIALS AND METHODS 2.1 Chemicals Sodium chloride (NaCl) was purchased from Merck KGaA, Darmstadt, Germany. Vegetable oils (palm and coconut oils) were purchased from Lam Soon (Thailand) Public Company Limited, Bangkok, Thailand. Xanthan gum was purchased from Deosen Biochemical (Ordos) Ltd, Shandong, China. 2.2 Extraction of allelopathic substants form mango and sugarcane leaves Mango and sugarcane leaves was obtained from Phitsanulok and Phichit (Thailand), it was grounded to 0.5–1 mm size and extracted with water at a ratio of 1:5. The mixture was left for 24 hours at a temperature of 4oC and then filtered through the thin white cloth followed by WhatmanTM filter paper (No. 1). The extracts from mango and sugarcane leaves were used as solution containing allelopathic substants. 2.3 Bacteria cultivation and biosurfactant Biosurfactants production from an alkaliphilic bacteria (Brevibacterium casei NK8) was performed according to [4] Briefly, coconut oil cake was used as substrate and cultured with Brevibacterium casei NK8 in the Horikoshi medium (pH 10) without glucose for biosurfactants production. After three days of cultivation, the production medium was separated from residues of fermented coconut oil cake and then centrifuged at 8,000 rpm (15 min) for cells removal. The obtained cell-free broth containing biosurfactants was used for bioherbicide formulation. 2.4 Emulsification of vegetable oils with cell-free broth containing biosurfactant For the preparation of emulsions, the mixture of 30% (v/v) cell free broth, 5% (w/v) NaCl, 0.1 % (w/v) xanthan gum, 2.5 % (v/v) vegetable oils (palm and coconut oils), 50 % (v/v) leaf extracts (mango and sugarcane leaves) and 12.4 % (v/v) DI water. The mixture was homogenized by vortex for 3 min and left for 14 days to observe the emulsion stability. The percentage of oil solubility in emulsion was calculate by followed equation: Oil solubility (%) = [volume of free oil (ml)/ volume of solubilized oil (ml)]x100 2.5 Seeds, plant growth, herbicide treatments, and spraying apparatus Bidens Pilosa L. seeds was collected from Phitsanulok (Thailand) and were seeded in soil trays (Peltracom, Belgium). When the seedlings were at the 3rd leaf stage after seeding. control Post-emergence herbicide was applied for 3 days once a day with 3 replicates at 1 ml per plant. When seedlings are 2 weeks old, they are transplanted into pots. When the weed seedlings were in the 3-5 leaf stage, they were sprayed with 1 ml of bioherbicide per plant. Plant damage was assessed visually compared to control plants at 1, 2, 3 and 14 days after spraying and the assessment that determines toxicity is based on applied concepts of the Brazilian Society of Weed Science (SBCPD), which describe the symptoms in detail (Table 1).

191 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Table 1 Description of concepts applied to toxicity assessments (Todero et al., 2018) 3. RESULTS AND DISCUSSION 3.1 Effect of vegetable oils on emulsification According to Table 2 and 3, solutions containing NaCl showed higher emulsification ability than no NaCl mixing. These results demonstrated the important role of NaCl on micelle formation with vegetable oils due to its hydrophobicity [2] The highest oil solubility was obtained from the mixture of 30 % cell-free broth, 2.5 % palm oil or coconut oil, 0.1 % xanthan gum, 50 % mango leaf extract, 5 % NaCl and 12.4 % DI water. This formula had highest mango leaf extract than others which might be resulting to the synergistic of their metabolites on oil solubilization in the core of micelle. [1] Moreover, the emulsion of this formula was stable for more than 1 month in room temperature. However, [6] formulated emulsion of lemongrass oil by sophorolipid and found the de-emulsion within 21 hours. The longer storage time of emulsion in this research might came from the encapsulation by polymer (xanthan gum) and polar compounds (NaCl and mango leaf extract). Table 2 Emulsion development of biosurfactant as a bioherbicide in Table 3 Emulsion development of biosurfactant as bioherbicide in combination with coconut oil 3.2 Effect of herbicidal activity on Bidens Pilosa L. To evaluate the potential of applying other extracts from plant leaves, mango-leaf emulsion was compared to sugarcane-leaf emulsion. The wilting of B. Pilosa was observed at 1, 2, 3 and 14 days and found that sugarcane-leaf emulsion showed the highest and rapid wilting within 24 hours followed by mango-leaf emulsion (Table 4). These results demonstrate that using all componentsin biosurfactant-based emulsion provided synergistic effect on herbicidal activity. However, all treatments show 80-100% plant damage which might be from the spraying solution to B. Pilosa on day 1, 2 and 3. Therefore, it has potential to reduce the frequent of spraying bioherbicide. [5] sprayed the mixture of Span80, Tween80 and palm oil on B. Pilosa and found the little symptoms (20-40%) of leaf discoloration after 48 hours of spraying. The developed bioherbicides was tested at sugarcane field in Nakhon Sawan (Thailand) with a size of 3 × 3 meters and the volume of spraying was 60 L/rai. Both mango-leaf emulsion and sugarcane-leaf emulsion had greater weed suppression effect than others (Table 5).

192 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Table 4 Phytotoxicity on plant of Bidens Pilosa L. the application of each formulation at 1,2,3 and 14 days

193 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Table 5 Test bioherbicide after spraying broad-leaf and narrow-leaf weeds in the sugarcane field for 1 day. 4. CONCLUSIONS The development of bioherbicides in the form of microemulsions using environmentally friendly ingredients has shown good weed suppression when tested in greenhouse conditions. Subsequently, these bioherbicides were tested in actual agricultural fields and found to effectively inhibit weed growth. Therefore, bioherbicides can reduce residual effects compared to chemical herbicides, while also being safe for users, the environment, and promoting sustainable agriculture. 5. ACKNOWLEDGEMENT The research team would like to thank the Department for funding from the National Research Council of Thailand (NRCT) for supporting the National Biological Control Research Center in the Lower Northern Region. Naresuan University, Phitsanulok, Thailand, in research under the project contract number N21A650767. 6. REFERENCES [1] Barreto, J. C., Trevisan, M. T., Hull, W. E., Erben, G., De Brito, E. S., Pfundstein, B., ... & Owen, R. W. (2008). Characterization and quantitation of polyphenolic compounds in bark, kernel, leaves, and peel of mango (Mangifera indica L.). Journal of agricultural and food chemistry, 56(14), pp. 5599-5610. [2] Cai, X., Du, X., Zhu, G., & Cao, C. (2020). Induction effect of NaCl on the formation and stability of emulsions stabilized by carboxymethyl starch/xanthan gum combinations. Food Hydrocolloids, 105, 105776. [3] Cordeau, S., Triolet, M., Wayman, S., Steinberg, C., & Guillemin, J. P. (2016). Bioherbicides: Dead in the water? A review of the existing products for integrated weed management. Crop protection, 87, pp. 44-49. [4] Khondee, N., Ruamyat, N., Luepromchai, E., Sikhao, K., & Hawangchu, Y. (2022). Bioconversion of lignocellulosic wastes to zwitterionic biosurfactants by an alkaliphilic bacterium: Process development and product characterization. Biomass and Bioenergy, 165, 106568. [5] Todero, I., Confortin, T. C., Luft, L., Brun, T., Ugalde, G. A., de Almeida, T. C., ... & Mazutti, M. A. (2018). Formulation of a bioherbicide with metabolites from Phoma sp. Scientia Horticulturae, 241, pp. 285-292. [6] Vaughn, S. F., Behle, R. W., Skory, C. D., Kurtzman, C. P., & Price, N. P. J. (2014). Utilization of sophorolipids as biosurfactants for postemergence herbicides. Crop Protection, 59, pp. 29-34.

194 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Sudthida Kamchonemenukool¹, Tipawan Thongsook¹, Panatpong Boonnoun² and Monthana Weerawatanakorn¹* 1 Department of Agro-Industry, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok, 65000, Thailand ²Department of Industrial Engineering, Chemical Engineering Program, Faculty of Engineering, Naresuan University, Phitsanulok 65000, Thailand *Correspondence to: [email protected] ABSTRACT: Sugarcane processing ofsugarmillsresultsinmany by-productsincluding sugarcane bagasse, filter mud (filter cake) and sugarcane leaves. These by-products cause the problem of pollution, but they are reported as a good source of cholesterol lowering nutraceuticals of policosanols. This study focused on using green technologies of supercritical fluid carbon dioxide and subcritical liquefied dimethyl ether extraction to extract policosanols from the by-products of sugar mills. The subcritical liquefied dimethyl ether extraction was operated at pressure of 1.0-1.5 MPa while supercritical fluid carbon dioxide extraction was operated at pressure of 20-27.5 MPa. The response surface methodology (RSM) with Box‑Behnken Design (BBD) was used to optimize the extraction conditions ofsupercritical fluid carbon dioxide extraction with three parameters including level of co-solvent as ethanol, temperature, and pressure to obtain the crude extract with a high policosanol content. By supercritical extraction, the results showed that the highest policosanol contents from filter mud (756.38 mg/100 g) was obtained at the co-solvent of 1 L, temperature of 70 °C, and pressure of 27.5 MPa. In contrast, one from sugarcane leaves (724.23 mg/100g) was obtained at the co-solvent of 1 L, temperature of 60 °C, and pressure of 20 MPa. The pressure has the greatest effect on policosanol content, followed by temperature and their interaction, while the volume of co-solvent slightly affected both materials. Compared with supercritical fluid carbon dioxide, the subcritical liquefied dimethyl ether extraction gave the high policosanol contents of 3,164.95 mg/100g for filter mud and 1,286.29 mg/100g for cane leaves. The study showed the potential use of the subcritical liquefied dimethyl ether as an alternative low-pressure green technique for fat-soluble functional ingredients of policosanols. Keywords: Filter mud, Sugarcane leaves, Policosanol, Supercritical carbon dioxide, Subcritical liquefied dimethyl ether P-017 Supercritical carbon dioxide and subcritical liquefied dimethyl ether extraction of policosanol from by-products of sugar mill

195 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Natcha Ruamyat¹, Khantharot Ditchat1, and Nichakorn Khondee1* 1 Department of Natural Resources and Environment, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Thailand *Correspondence to: Department of Natural Resource and Environment, Faculty of Agriculture, Natural Resource and Environment, 99 Moo 9 Naresuan University, Phitsanulok, 65000, Thailand, E-mail: [email protected] ABSTRACT: Nowadays, sugar is in great demand as a flavoring for foodstuffs. A large amount of by-products produced from the sugarcane industry are yeast sludge and sugarcane filter cake. To utilize and increase the value of these by-products, the purpose of this research is to develop a biological process by employing alkaliphilic Brevibacterium casei NK8 to convert by-products from the sugarcane industry into biosurfactants. The optimal conditions of biosurfactant production were determined using central composite design (CCD) by setting yeast sludge and sugarcane filter cake in a range of 1-5% and 2-10%, respectively. The results from the statistical analysis of response surface methodology (RSM) showed that biosurfactant was highly produced. To evaluate the potential of the produced biosurfactants as agricultural enhancement and industrial plant cleaning agents, the contact angles of biosurfactants on various plant leaves and materials were measured, such as glass slides, stainless steel, parafilm, T. procumbens, B. oleracea, and sugarcane leaves. The results showed that biosurfactants had higher wettability than commercial biosurfactants (rhamnolipid and sophorolipid) and chemical surfactants (Tween 80). Therefore, utilizing by-products generated from the sugarcane industry as substrates for the production of biosurfactants is a sustainable waste management and value-adding strategy. The produced biosurfactants are able to improve the efficiency of agrochemicals and fertilizersin crops and clean hard surfacesin industrial plants(machines, glass, and packaging) safely. Keyword: Sugarcane by-products, Alkaliphilic Brevibacterium casei NK8, Statistical optimization, Biosurfactants, Sustainable waste management P-018 Sustainable production of biosurfactants via bioconversion of by-products from the sugarcane industry by alkaliphilic Brevibacterium casei NK8 for crop enhancement and industrial cleaning applications: a statistical optimization by CCD-RSM

196 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS 1. INTRODUCTION The world's agro-based economic sector is primarily supported by the sugar industry. A valuable cash crop, sugarcane is utilized as a raw material in the sugar industry. In 2021, the average worldwide sugarcane production was over 0.7 million hectograms per hectare, which accounted for approximately 80% of the world's sugar production [1]. This indicates their essential economic significance by showing how easily they can be acquired for a cheap price and in huge quantities [2]. Sugarcane waste includes products such as sugarcane stalks, bagasse, molasses, and straw that are produced during the sugarcane processing process. Still, there are seven phases involved in the processing of sugarcane: harvesting, cleaning, chopping, extracting the juice, purifying, evaporating, and crystallizing [3]. Furthermore, sugarcane wastes generally have a high quantity of nutrients, including cellulose, hemicellulose, lignin, minerals, amino acids, etc., making them suitable as substrates for the production of biosurfactants. Agro-industrial waste management can potentially be considered more sustainable via the bioconversion of lignocellulosic wastes to biosurfactants [4]. The advantages of biosurfactants include several compared to chemical surfactants, such as their biodegradability, biocompatibility, extended foaming activities, high selectivity, and effectiveness in highly toxic environments [5]. Weakness in the production of biosurfactants at large scale is caused by pricey raw materials, a low production yield, a difficult downstream stream process, and a high purifying cost. In most biotechnological procedures, it is assumed that ingredients (production medium) contribute 10–30% of the entire production costs. The expense associated with producing microbial biosurfactants, mostly as a result of expensive substrates, is the primary factor limiting their application [6]. Khondee et al. [7] have investigated several lignocellulosic wastes, including sugarcane bagasse, wheatstraw,Jatropha cake, rice husk, rice straw, maize cobs, and woodchips, for the production of biosurfactants. The materials can be pretreated using a variety of methods, including enzyme hydrolysis, acid hydrolysis, alkali pretreatment, and hydrothermal treatment. The combined alkaline and hydrothermal pretreatment had low inhibitors released from lignocellulose degradation [8]. Khondee et al. [7] show that an alkaliphilic bacteria utilized both liquid and solid from the combined alkaline and hydrothermal pretreatment as substrates for biosurfactant production without a pH neutralization process. The objective of this research was to utilize by-products (sugarcane filter cake and yeast sludge) from the sugarcane industry for biosurfactant production by alkaliphilic bacteria and to investigate the potential application of the produced biosurfactant by their surface activities with various materials. This research was divided into two phases: 1) statistical optimization of biosurfactant production using a mix of sugarcane filter cake and yeast sludge by central composite design (CCD) and respond surface methodology (RSM); and 2) determination of contact angle reduction of cell-free broth with and without lignin on a glass slide, stainless steel, parafilm, T. pocumbens, B. oleracea, and sugarcane leaf compared with commercial biosurfactants and chemical surfactants. 2. MATERIALS AND METHODS 2.1 Materials and biosurfactant-producing bacteria The sugarcane filter cake and yeastsludge were collected from the sugarcane industry in Thailand and used asthe primary sources of carbon and nitrogen for the bacteria's growth and biosurfactant production. They were sterilized and dried in a hot air oven at 60 °C. The alkaliphilic biosurfactant-producing organism identified as Brevibacterium casei NK8 was discovered in an alkaline-contaminated soil sample collected from a factory in Thailand that refined vegetable oil. Brevibacterium casei NK8 was cultivated in Horikoshi broth (pH 10, 10 g L-1 glucose, 5 g L-1 peptone, 5 g L-1 yeast extract, 1g L-1 K2HPO4, 0.2 g L-1 MgSO4•7H2O, 10 g L-1 Na2CO3). They were sub-cultured in Horikoshi broth at 30 °C for 72 hours after being stored at -4°C in 50% glycerol stocks. 2.2 Pretreatment and fermentation of mixed sugarcane filter cake and yeast sludge for biosurfactant production Sugarcane filter cake (carbon source) and yeast sludge (nitrogen source) were mixed in a 250-mL Erlenmeyer flask, and a 10% (w/v) Na2CO3 solution was applied as a substitute for Horikoshi medium to produce biosurfactant. Bacterial inoculum was prepared by adding 1 ml of bacterial stocks to the broth and incubating for 72 hours at 30 °C and 200 rpm. The mixed residues were autoclaved at 110 °C for 10 minutes. After allowing the autoclaved medium to cool to room temperature without adjusting the pH, 10% (v/v) bacterial inoculum (OD600 = 1) was added to begin the fermentation process. To produce biosurfactant, the culture was incubated at 30°C and 200 rpm for 96 hours. The number of Brevibacterium casei NK8 was analyzed at 0 and 96 hours. After fermentation, a sterile 300 μm stainless steel filter was used to separate the sterile production medium from the residual solid substrate. The filtered production medium was centrifuged at 8,000 rpm for 30 minutes. Cell-free broth without lignin was prepared by adjusting the pH of cell-free broth to 4.0 using 6 M HCl [9]. After the lignin was removed, biosurfactants in cell-free broth were determined using organic solvent extraction techniques [10]. The contact angle of cell-free broth with and without lignin was measured on sugarcane leaf, weed leaf, vegetables, stainless steel, and glass by an optical contact angle instrument (Dataphysics, OCA20).

197 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS 2.3 Experimental design for biosurfactant production using CCD-RSM CCD-RSM was applied for this research to achieve the highest biosurfactant production. As independent variables (factors), sugarcane filter cake and yeast sludge were varied in the range of 2–10% (w/v) and 1–5% (w/v), respectively. The dependent variables (responses) were biosurfactant concentration, bacterial growth, lignin concentration, and contact angle. The accuracy of this model (using the STATISTICA software version 8.0) was shown by the observation that the experimentally determined actual values closely corresponded to the predicted values provided by the CCD-RSM design. The highest concentration of biosurfactant (g/L) demonstratesthe optimum combination of process variables. Optimization design responses were evaluated using the analysis of variance (ANOVA). The interaction effects and significance of the variables influencing the production of biosurfactants were investigated using ANOVA. The consequent values for the R2 coefficient of determination and the R2 adjusted value were used to evaluate the polynomial. The model significance corresponds to the probability "p" function. Under optimal conditions, the obtained model's validity was experimentally (in triplicate) verified. 3. RESULTS AND DISCUSSION 3.1 ANOVA and RSM analyses for the production of biosurfactant using mixed sugarcane filter cake and yeast sludge CCD was performed to evaluate the optimum level, influence, and interactions of two factors, such as sugarcane filter cake (A) and yeast sludge (B), on bacterial number, biosurfactant concentration, and lignin concentration. The factors actual and coded values are shown in Table 1 at three coded levels(-1, 0 and + 1). Table 1 represented the design matrix for the 10 experiments. (Table 2). When the variance of the independent variable had the highest R-square value of each dependent variable, i.e., bacterial number had an R2 value of 0.9791, biosurfactant concentration had an R2 value of 0.9270, and lignin concentration had an R2 value of 0.9974, this was selected as the suitable model type. Table 3 demonstrates the empirical coefficient of each term in equations to predict bacterial number (Y1, log10CFU/ ml), biosurfactant concentration (Y2, g/L), and lignin concentration (Y3, g/L). Sugarcane filter cake and yeast sludge had significant effects on bacterial growth (P-value < 0.05). Table 1 CCD-RSM experimental design matrix of significant process parameters for bacterial number, biosurfactant concentration and lignin concentration

198 The 2nd International Conference on Cane and Sugar 2023 Towards BCG Economy; Smart Farm to Bio Industry CO-PRODUCTS Table 2 Regression coefficient and analysis of variance for the quadratic model Linear/Quadratic main eff. +2-way from the CCD-RSM model Table 3 ANOVA analysis for bacterial number, biosurfactant concentration and lignin concentration from CCD-RSM model a Confident of interval = 95%, Alpha = 0.05 3.2 Influence of process variables on bacterial growth, biosurfactant concentration and lignin concentration By graphing the response functions of two independent variables while keeping the other at the center point, 3D response surface plots were created to determine the optimum level of each independent component for optimal production.According to Figure 1, high bacterial growth and biosurfactant concentrations were found in both low and high sugarcane filter cakes. High bacterial growth was found from moderate yeast sludge, while high biosurfactant concentration was achieved from both low and high sugarcane filter cake. These phenomena could be caused by the C/N ratio. One of the most important factors influencing microbial growth and biosurfactant production during fermentation processes was the C/N ratio. Lower nitrogen concentrations (i.e., high C/N ratios) inhibited bacterial growth, thus supporting cellular metabolism towards the synthesis of biosurfactant [11]. The optimal mixtures of sugarcane filter cake and yeast sludge were 14 and 7% (w/v), respectively. Under this condition, concentrations of biosurfactant and lignin were 2.09 and 1.74 g/L, respectively, while bacterial numbers were 8.35 log10 CFU/ml. Jain et al. [12] found that biosurfactant production by Klebsiella sp. RJ-03 using rice husk and wheat straw as substrates produced biosurfactants of 0.94 and 1.03 g/L.