Patent Application: US-10297608-A

Abstract:
disclosed are processes for liquefaction and saccharification of polysacharide containing biomasses having high dry matter content and preferably possessing large average particle size . the polysaccharide containing biomasses are subject to enzymatic hydrolysis in a mixer that utilizes “ free fall ” mixing . “ free fall ” mixing provides mechanical degradation of the biomass during hydrolysis and , in the case of lignocellulosic biomass , promotes movement of cellulase enzymes along cellulose chains , which improves enzymatic hydrolysis at high dry matter .

Description:
production of concentrated sugar solutions is beneficial in relation to subsequent fermentation or other microbial processes due to improved volumetric productivity and reduced cost of down stream processing . in case of bio - ethanol production , the energy requirement for distillation is significantly reduced if the fermentation broth contains above 4 % ethanol ( galbe and zacchi , 2002 ). this requires a sugar concentration above 8 %, which with most types of lignocellulosic biomasses corresponds to an initial dry matter content above 20 %. it is in other words desirable to subject polysaccharide - containing biomasses with high dry matter contents , preferably above 20 %, to enzymatic hydrolysis in order to be able to subsequently produce bio - ethanol - containing fermentation broths suitable for distillation of ethanol . the processes of the present invention provide a degree of enzymatic hydrolysis of typically 30 - 50 %. however , under optimised conditions even a higher degree of enzymatic hydrolysis may be obtained . the liquefied and saccharificated biomass will consequently contain relatively large amounts of glucose , xylose , cellobiose , lignin , non - degraded cellulose and hemicellulose and still active enzymes suitable for further processing i . e . fermentation processes ( ethanol , lactic acid etc .). the liquefied biomass will also be suitable for gasification , hydrogenation , organic synthesis , or production of biogas and feed . if the polysaccharide containing biomasses are lignocellulosic the pre - treatment must ensure that the structure of the lignocellulosic content is rendered more accessible to the enzymes , and at the same time the concentrations of harmful inhibitory by - products such as acetic acid , furfural and hydroxymethyl furfural remain substantially low . there are several strategies to achieve this , which all implies subjecting the lignocellulosic material to temperatures between 110 - 250 ° c . for 1 - 60 min e . g . : hot water extraction multi stage dilute acid hydrolysis , which removes dissolved material before inhibitory substances are formed dilute acid hydrolyses at relatively low severity conditions alkaline wet oxidation steam explosion almost any pre - treatment with subsequent detoxification polysaccharide containing biomasses according to the present invention includes any material containing polymeric sugars e . g . in the form of starch as well as refined starch , cellulose and hemicellulose . biomasses having a dry matter content above 20 % are preferred . relevant types of biomasses for enzymatic hydrolysis and mixing according to the present invention may include biomasses derived from agricultural crops such as e . g . : starch e . g . starch containing grains and refined starch corn stover bagasse straw e . g . from rice , wheat , rye , oat , barley , rye , rape , sorghum softwood e . g . pinus sylvestris , pinus radiata hardwood e . g . salix spp . eucalyptus spp . tubers e . g . beet , potato cereals from e . g . rice , wheat , rye , oat , barley , rye , rape , sorghum and corn waste paper , fibre fractions from biogas processing , manure , residues from oil palm processing , municipal solid waste or the like with a similar dry matter content . if the polysaccharide containing biomasses are lignocellulosic , the material may be cut into pieces where 20 % ( w / w ) of the biomass preferably ranges within 26 - 70 mm , before pre - treatment . the pre - treated material has preferably a dry matter content above 20 % before entering the mixing device . besides liberating the carbohydrates from the biomass , the pre - treatment process sterilises and partly dissolves the biomass and at the same time washes out potassium chloride from the lignin fraction . the mixing performed in a process according to the present invention serves at least a four - fold purpose . firstly , it ensures close contact between the enzymes used and the polysaccharide containing biomass ( substrate ), as this will in most cases be insoluble or only very slightly soluble . secondly , the mechanical work performed on the material during the mixing helps tearing larger biomass fibres and particles apart and will therefore assist in increasing the surface area of the material . this will increase the accessibility of e . g . cellulose and hemicellulose to the enzymes used . to further increase the mechanical work on the material , steel balls or similar means that will collide with the material might be added to the drum . thirdly , the mixing of the material prevents local accumulation of high cellobiose concentration that — as is well known for a person skilled in the art — can inhibit e . g . cellulase enzymes , especially the cellobiohydrolases . fourthly , an important characteristic of the cellulase enzymes is the influence of cellulose binding domains ( cbd ) on the enzyme performance . cbd &# 39 ; s are functional parts of cellulose degrading enzymes . the cbd enables adhesion of the water - soluble enzyme onto an insoluble substrate surface ( cellulose ). the close association between the enzyme and cellulose provided by the cbd enhances the catalytic rate and stability of the enzyme . to hydrolyse cellulose , the enzyme must change the position of the cbd on the cellulose chain . it is believed that mechanical action , i . e . mixing , is important for the movement of the cbd and consequently for the enzymatic action of the enzymes along the cellulose chain . in addition to the above it should be noted that enzymatic hydrolysis of biomass has traditionally been conducted in stirred tank reactors equipped with impellers ( e . g . rushton turbine or intemig impeller ) mounted on a centrally placed impeller shaft similar to what is used in the fermentation industry . due to this equipment , solutions of high viscosity , very sticky or very dry material cannot be stirred efficiently but will result in zones with very poor or no mixing . furthermore , stirrings of such solutions require very large energy inputs , which is detrimental to the process economy . operating with polysaccharide containing biomasses this has previously restricted the upper possible limit to app . 20 %. the gravity based mixing principle according to the present invention overcomes this problem and may be used for polysaccharide containing biomasses with a dry matter content up to 80 %, preferably 20 - 50 %. the principle of gravity mixing according to the present invention can easily be scaled up and be applied for all kinds of biomasses , besides refined starch , containing up to more than 80 % cellulose . unlike conventional stirred tank reactors traditionally used for enzymatic hydrolysis , a gravity based mixing principle , i . e . a drum mixer , a mixer with a rotary axis lifting the biomass or similar mixing devise utilising a free fall principle , at the same time enables an efficient mixing even with small power inputs and high dry matter contents and furthermore performs a mechanical processing / degradation through the forces of gravity including shear and tear forces between material and drum as well as the forces resulting from the impact between falling material and the bottom of the drum and at the same time positively effects the influence of cellulose binding domains ( cbd ) on enzyme performance . although processing of non - miscible plant materials , such as e . g . polysaccharide containing biomass with relatively high dry matter content and large average fibre and particle size , is known from solid - state fermentation or bioreactors , where tumble type mixers are used for blending ( giovanozzi et al . 2002 ), this principle has not previously been implemented in a dedicated liquefaction / saccharification process or a bio - ethanol fermentation process . the present invention provides a process for processing of biomasses at relatively high dry matter contents , e . g . dry matter contents between 20 - 80 %, preferably between 20 - 50 %. furthermore , the process according to the present invention ensures efficient liquefaction and saccharification enabling the direct use of the end product in e . g . fermentors . enzymes capable of effecting a conversion of starch , cellulose and hemicellulose or parts thereof into glucose , xylose and cellobiose are added to the biomass either in native form or in form of microbial organisms giving rise to the accumulation of such enzymes . the ph and the temperature of the biomass are adjusted with reference to the ph - optimum and the temperature optimum of the enzymes applied . depending on enzyme loading , the biomass will be liquefied and saccharified to a liquid without any or only with few remaining large fibres and particles within 3 - 24 hours . adding a glucose metabolising microorganism at any given time during the hydrolysis and liquefaction may enhance the degree of enzymatic hydrolysis as inhibitory enzyme products are thereby removed . a process according to the present invention can be performed using the following preferred technical parameters . dry matter content : 20 - 80 %, preferably 25 - 70 %, more preferably 25 - 60 %, even more preferably 25 - 50 % or 25 - 40 % and most preferably 25 - 35 % distribution of fibre and particle sizes of lignocellulosic biomass : 0 - 150 mm , preferably , 5 - 125 mm , more preferably , 10 - 100 mm , even more preferably 15 - 90 mm or 20 - 80 mm and most preferably 26 - 70 mm . the preferred distribution of fibre and particle sizes is defined as at least 20 % ( w / w ) of the lignocellulosic biomass ranging within the preferred interval . if the polysaccharide containing biomass is lignocellulosic , it has to be pre - treated e . g . by a hot water extraction . if a hydro thermal pre - treatment is chosen the following technical data are preferred : pre - treatment temperature : 110 - 250 ° c ., preferably 120 - 240 ° c ., more preferably 130 - 230 ° c ., more preferably 140 - 220 ° c ., more preferably 150 - 210 ° c ., more preferably 160 - 200 ° c ., even more preferably 170 - 200 ° c . or most preferably 180 - 200 ° c . pre - treatment time : 1 - 60 min , preferably 2 - 55 min , more preferably 3 - 50 min , more preferably 4 - 45 min , more preferably 540 min , more preferably 5 - 35 min , more preferably 5 - 30 min , more preferably 5 - 25 min , more preferably 5 - 20 min and most preferably 5 - 15 min dry matter content after pre - treatment of at least 20 w / w %. if a vessel based on the free fall mixing concept in the form of a reactor with a horizontal placed stirrer shaft lifting the biomass or similar mixing devise is used , the following technical data are preferred : rotational speed : 0 - 30 rpm , preferably 0 - 20 rpm , more preferably 0 - 15 rpm even more preferably 0 - 10 rpm and most preferably 0 - 5 rpm . rotation with periodically alternated rotating direction . rotation in pre - defined intervals . the optimal rotational speed will depend on the volume of the vessel , the preferred rotational speed may thus be relatively high when the process is carried out in a relatively small vessel , while it may be relatively low when the process is carried out in a relatively large vessel . enzyme loading in filter paper units ( fpu )/ g dm . 1 fpu equals the amount of enzyme necessary to hydrolyse 1 μmol / min of glycosidic bonds on whatmann # 1 filter paper , under specified conditions well known to a person skilled in the art . however , enzymatic activity could in principle be supplied in any conceivable form including through the addition of microorganisms giving rise to the desired enzymatic activity : corresponding to 0 . 001 - 15 fpu / g dry matter , preferably 0 . 01 - 10 fpu / g dry matter , more preferably 0 . 1 - 8 fpu / g dry matter , more preferably 1 - 7 fpu / g dry matter and most preferably less than 6 fpu / g enzymes for starch containing biomass : treatment time for enzymatic hydrolysis : 0 - 72 hours , preferably 1 - 60 hours , more preferably 2 - 48 hours and more preferably 3 - 24 hours such as 4 - 24 hours , such as 6 - 24 hours , such as 8 - 24 hours , such as 10 - 24 , such as 12 - 24 hours , such as 18 - 24 hours or 22 hours temperature for enzymatic hydrolysis . adjusted with reference to the optimum temperatures of the applied enzymatic activities : 0 - 105 ° c ., preferably 10 - 100 ° c ., more preferably 15 - 90 ° c ., more preferably 20 - 80 ° c ., more preferably 25 - 70 ° c . and most preferably 30 - 70 ° c . such as 40 - 45 ° c . or room temperature . ph of biomass . adjusted with reference to the optimum ph of the applied enzymatic activities : 3 - 12 , such as 5 - 10 , such as 6 - 9 , such as 7 - 8 and preferably 4 - 11 the enzymatic treatment can be conducted as a batch , fed batch or a continuous process . pressed pre - treated wheat straw with an average size of approximately 40 mm ( counter - current water extraction at 180 - 200 ° c . for 5 - 10 min ., water and dry matter flow ratio of 5 : 1 ) corresponding to 25 g dry weight (= 67 . 0 g pre - treated straw ) was put into a plastic bag . 0 . 75 ml of novozym 188 , 3 . 75 ml of celluclast 1 . 5 fg l and 11 . 9 ml of 50 mm sodium citrate buffer , ph 5 . 0 , was mixed and sprayed onto the straw . this resulted in a final dry matter content of 30 %. the enzyme loading corresponded to 10 filter paper units ( fpu )/ g dm . the mixer consisted of a drum ( 1 . 0 m long and 0 . 78 m in diameter ) with 5 internal ribs along the long axis to ensure proper mixing of the material . the drum rotated along the horizontal axis with a speed of 26 rpm . the mixing / hydrolysis of the material was performed for 18 - 24 hours at room temperature . this resulted in a thick paste without any remaining large fibres . a control bag with the same enzyme loading but no mixing showed no sign of degradation of the straw . part of the resulting material after the enzymatic hydrolysis for 24 hours ( an amount corresponding to 29 g dry matter ) was diluted to 15 % dry matter in a blue cap bottle and yeast ( baker &# 39 ; s yeast , de danske spriffabrikker ) was added . the bottle was closed by an air lock and placed for 72 hours at 30 ° c . with stirring at 500 rpm . the resulting liquid contained 33 g / l of ethanol , 10 g / l of xylose . no glucose was detected indicating that the yeast was capable of utilising all glucose produced during the hydrolysis . assuming an ethanol yield on glucose of 0 . 5 g ethanol per 9 glucose this corresponded to conversion of 70 % of the originally cellulose . pressed pre - treated wheat straw with an average size of approximately 40 mm ( pre - treated by counter - current water extraction at 180 - 200 ° c . for 5 - 10 min . with a water and dry matter flow ratio of 5 : 1 ) corresponding to 7 kg dw (= 20 kg pre - treated straw ) was put into a conventional rotary cement mixer , with a horizontal axis tilted about 10 degrees . the mixer had 2 internal ribs along the long axis to ensure mixing of the material . a lid was mounted on the opening to avoid evaporation from the mixer . the mixer drum rotated along the horizontal axis with a speed of 29 rpm . 200 - 1150 ml of celluclast 1 . 5 fg l and 40 - 225 ml of novozym 188 were added to the straw . this resulted in a final dry matter content of 30 %. the enzyme loading corresponded to 3 - 15 fpu / g dm . the ph was adjusted to 4 . 8 to 5 . 0 by addition of sodium carbonate . the cement mixer was heated to 40 - 45 ° c . by use of a fan heater . the mixing / hydrolysis of the material was performed for 22 hours . depending on enzyme loading this resulted in a more or less viscous liquid without any remaining large fibres . the pre - treated straw was degraded to a paste in app . 3 - 5 hours . after 5 - 24 hours of mixing the paste was changed to a viscous liquid . control experiments with pre - treated wheat straw only or wheat straw pre - treated at only 160 ° c . but using the same enzyme loading showed no sign of liquefaction of the straw . simultaneous saccharification and fermentation was performed by adding yeast to the cement mixer after 24 hours of hydrolysis at 40 - 45 ° c . using an enzyme loading of 10 - 15 fpu / g dm . the temperature was allowed to cool to below 35 ° c . and compressed yeast ( baker &# 39 ; s yeast , de danske spriffabrikker ) was added to a concentration of approximately 1 % ( w / w ) based on initial dry matter of straw . the saccharification and fermentation was continued for 48 hours at 25 ° c . the resulting material was centrifuged for 15 min at 2500 rpm . the supernatant was filtered through a 0 . 45 μm filter and analysed for sugars on hplc . at an enzyme load of 15 fpu / g dm , the supernatant contained 70 g / l of glucose , 30 g / l of xylose after 24 hours of hydrolysis . this corresponded to 50 % hydrolysis of the cellulose and hemicellulose originally present in the straw . the simultaneous saccharification and fermentation using an enzyme loading of 10 fpu / g dm resulted in 42 g / l of ethanol and 30 g / l of xylose . the hydrolysis reactor was designed in order to perform experiments with liquefaction and hydrolysis solid concentrations above 20 % dm shown in longitudinal view in fig1 a and in transverse view in fig1 b . the reactor consisted of a horizontally placed drum divided into 5 separate chambers each 20 cm wide and 60 cm in diameter . a horizontal rotating shaft mounted with three paddlers in each chamber was used for mixing / agitation . a 1 . 1 kw motor was used as drive and the rotational speed was adjustable within the range of 2 . 5 and 16 . 5 rpm . the direction of rotation was programmed to shift twice a minute between clock and anti - clock wise . a water - filled heating jacket on the outside enabled the control of the temperature up to 80 ° c . the chambers were filled with pressed pretreated wheat straw with an average size of approximately 40 mm ( pre - treated by counter - current water extraction at 180 - 200 ° c . for 5 - 10 min . with a water and dry matter flow ratio of 5 : 1 ) and water to give an initial dm content of 20 to 40 %. celluclast 1 . 5 fg l and novozym 188 in 5 : 1 ratio were added to give an enzyme loading of 7 fpu per g dm . the liquefaction and hydrolysis was performed at 50 ° c . and ph 4 . 8 to 5 . 0 . the mixing speed was 6 . 6 rpm . simultaneous saccharification and fermentation ( ssf ) experiments were performed by lowering the temperature to 32 ° c . after 8 h of liquefaction and hydrolysis and following the addition of 15 g of compressed baker &# 39 ; s yeast ( de danske spriffabrikker ) per kg of initial dm . liquefaction and hydrolysis was possible with initial dm content of up to 40 % dm ( see fig2 and fig3 ). with initial 40 % dm it was possible to reach glucose concentrations of 80 g kg − 1 after 96 h . it was also possible to operate the process as ssf ( see fig3 ), thereby reducing the product inhibition of the cellulases caused by the glucose accumulation . it was possible to ferment the hydrolysates with up to 40 % initial dm content using normal baker &# 39 ; s yeast . under not fully anaerobic conditions the ethanol yield was 80 , 79 , 76 , 73 and 68 % of what was theoretically obtainable at 20 , 25 , 30 , 35 and 40 % dm , respectively . lignocellulosic and starch containing biomass can be processed simultaneously using gravity mixing and a mixture of cellulases , hemicellulases and amylases . the lignocellulosic biomasses may be derived from agricultural crops consisting of e . g . corn stover , straw e . g . from rice , wheat , rye , oat , barley , rye , rape and sorghum , tubers e . g . beet , potato , cereals from e . g . rice , wheat , rye , oat , barley , rye , rape , sorghum , wood consisting of softwood e . g . pinus sylvestns , pinus radiata , hardwood e . g . salix spp ., eucalyptus spp ., municipal solid waste , waste paper and similar biomasses . the hydrolysis reactor described in example 3 was used for the experiments . wheat straw ( primarily a lignocellulose source ) was pretreated using counter - current water extraction at 180 - 200 ° c . for 5 - 10 min . with a water and dry matter flow ratio of 5 : 1 . wheat grain ( primarily a starch source ) was dry milled using a kongskilde roller mill . the wheat grain and pretreated straw with an average size of approximately 40 mm was mixed in a 1 : 1 ratio on a dry basis . dm was adjusted to between 30 and 40 % by addition of water . celluclast 1 . 5 fg l and novozym 188 in a 5 : 1 ratio were added to give an enzyme loading of 7 fpu per g dm of straw . hydrolysis of starch was carried out using cold mash enzyme ns50033 ( novozymes a / s , bagsværd , denmark ) at a loading of 3 . 5 g per kg of wheat grain . the liquefaction and hydrolysis was performed at 50 ° c . and ph 4 . 8 to 5 . 0 . after 8 h , the temperature was lowered to 34 ° c . and 15 g of compressed baker &# 39 ; s yeast ( de danske spritfabrikker ) was added per kg of initial dm . an experiment with straw only at 30 % dm was run in parallel . mixing straw with grain resulted in a fast initial accumulation of glucose in the liquefaction and hydrolysis step compared to applying straw only ( fig4 ). after 96 h of liquefaction and ssf the ethanol concentration was 41 g kg − 1 using only wheat straw as the only substrate ( see fig4 ). in the experiment with straw and grain the ethanol concentration reached 68 g kg − 1 . a process according to the present invention can also be applied for low temperature processing of refined starch or starch containing materials ( e . g . beet , potato , cereals from e . g . rice , wheat , rye , oat , barley , rye , sorghum ). according to example 4 , heat pre - treatment of the grain is not necessary for liquefaction and hydrolysis of starch . dry milling is on the other hand generally used for pre - treatment of starch containing grains . dry milled grains with a dry matter content of 20 - 60 % are loaded into the gravity mixer . cold mash enzyme ns50033 ( novozymes a / s , bagsværd , denmark ) or alpha - amylase and glucoamylases are added simultaneously . a full liquefaction and saccharification of the starch is then possible in a one - pot process . temperature and ph ranges during the enzymatic hydrolysis process are defined by the enzymes and will be in the range of 25 - 60 ° c ., preferably 40 - 55 ° c ., and ph 3 - 12 , preferably ph 3 - 8 , respectively . galbe , m ., zacchi , g . 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