Patent Application: US-72963207-A

Abstract:
a process for the conversion of monocot lignocellulosic grass waste from mushroom growth into byproducts is described . in particular , the present invention releases glucans from the waste which can be easily hydrolyzed , after a less severe thermochemical process , and into sugars for producing ethanol or other by - products by fermentation .

Description:
rice straw ( rs ) is an important lignocellulosic biomass with nearly 900 million dry tons produced annually worldwide . rice straw has great potential as lignocellulosic feedstock for making renewable fuels and chemicals in a biorefinery . however , due to its natural recalcitrance , rice straw needs thermochemical treatment prior to further biological processing . ammonia fiber expansion ( afex ) is a leading biomass pretreatment process utilizing concentrated / liquefied ammonia to pretreat lignocellulosic biomass at moderate temperatures ( 70 - 120 ° c .). previous research has shown improved cellulose and hemicellulose conversions upon afex treatment of rice straw at 2 : 1 ammonia to biomass ( w / w ) loading , 40 % moisture ( dwb ) and 90 ° c . however , there is still room for much improvement . fungal pretreatment of lignocellulosics is an important biological pretreatment method that has not received much attention in the past . this is probably due to the long residence time of this process and also reduces overall yield . however , the overall sugar loss may be minimized through use of white - rot fungi ( i . e . pleutorus ostreatus ) over a much shorter incubation time , if combined with a subsequent low severity thermochemical pretreatment step . it was found that mushroom spent straw ( mss ) with afex allowed reduction in pretreatment severity , while giving higher glucan conversions ( up to 25 %) than before . in this invention , the effect of fungal conditioning of rice straw on afex pretreatment and enzymatic hydrolysis is described . the recovery of other byproducts from the fungal pretreatment process such as fungal enzymes , mushrooms , oligosaccharides , organic acids , and the like are also disclosed . untreated and afex treated biomass were presoaked and washed in distilled ( de - ionized ) water with a substrate to water loading of 1 : 10 ( w / w ). the slurry was mixed for 15 minutes . the wash liquid was removed from the substrate by squeezing the slurry through a filtration cloth ( calbiochem , ca ) and stored in the refrigerator for further analysis . the washed substrates were enzymatically hydrolyzed immediately to prevent microbial growth . the moisture content of the washed substrate was determined using a moisture analyzer ( model mf - 50 , a & amp ; d ). a mass balance for determining the solids and soluble content of the wash liquid stream was carried out according to the procedure outlined before . untreated rice straw ( rs ) and mushroom spent straw ( mss ) ( 200 gms dry bm each ) were soaked and stirred in distilled ( de - ionized ) water with a substrate to water loading of 1 : 10 for 15 minutes . the wash liquid was removed from the substrate by filtering the slurry through a filtration cloth ( calbiochem , ca ) and centrifuged at 9000 rpm to remove fine solid particles from the wash stream , then concentrated using membrane filtration ( 10 kda ). the supernatant was lyophilized to recover the washed soluble from the biomass for further characterization . the moisture content of the sample was determined after keeping the sample for 12 h in a lab oven at 105 ° c . lignin , sugar and protein content in the biomass were measured by dairy one ( new york , ithaca ) using standard analytical protocols . the composition analysis were done for rice straw ( rs ) and mushroom spent straw ( mss ) using nrel protocol ( lap001 , 002 and 012 ) using dilute acid followed by hplc analysis as given below . mushroom spent straw ( mss ) and rice straw ( rs ) were pretreated by the afex pretreatment process . the biomass with varying moisture ( 10 - 60 %) ( kg water / kg dry biomass ) was transferred to a high - pressure parr reactor and liquid ammonia ( 1 kg of ammonia / kg of dry biomass ) was slowly charged to the vessel . the temperature was raised and maintained at 90 ° c . for five minutes residence time at that temperature before explosively relieving the pressure . overall , it took approximately 30 - 40 minutes to complete one cycle of pretreatment process . the instantaneous drop of pressure in the vessel caused the ammonia to vaporize , causing an immediately decompression of the biomass and considerable fiber disruption . the pretreated material was allowed to stand under a hood overnight to remove the residual ammonia and stored in a freezer until further use . the nrel standard protocol ( lap - 009 ) was followed for enzymatic hydrolysis of the biomass . cellulase ( spezyme cp ) enzyme was a generous gift from genencor international ( rochester , n . y .). the substrate was hydrolyzed at a glucan loading of 1 % ( w : v ) in a 0 . 05 molar citrate buffer solution ( ph 4 . 8 ) at the desired cellulase enzyme loading ( protein concentration 123 mg / ml ) of 15 fpu / gm glucan and β - glucosidase ( sigma , st . louis , mo .) loading of 64 pnpgu / gm glucan . xylanase ( protein concentration 42 mg / ml ) supplementation was carried out at 10 % of the total milligrams of cellulase protein loaded . the protein concentration of the enzymes was determined by the bca protein assay ( pierce , rockford , ill .). samples were hydrolyzed at 50 ° c . with gentle agitation ( 90 rpm ) for a period of 168 hours . the hydrolyzed samples were boiled to denature the enzymes and filtered through a 0 . 2 micron nylon membrane filter at predetermined time periods ( 72 and 168 hours ). the samples were frozen for subsequent hplc sugar analysis . a high performance liquid chromatography ( hplc ) system was used for sugar analysis . the hplc system consisted of waters ( milford , mass .) pump and waters 410 refractive index detector , an aminex hpx - 87p carbohydrate analysis column ( biorad , hercules , ca ) equipped with a de - ashing guard cartridge ( biorad ). degassed hplc grade water was used as the mobile phase at 0 . 6 ml / min at a column temperature of 85 ° c . the injection volume was 20 μl with a run time of 20 min . mixed sugar standards were used for quantification of cellobiose and other monosaccharides ( glucose , xylose , galactose , arabinose and mannose ) in the samples . with the growing demand for ethanol , biorefinery using lignocellulosic feed stock can be successfully implemented depending on its large scale availability of biomass on a regular basis . since corn starch and cane sugar ethanol plants are well established , lignocellulosic waste generated from these plants can as well be transported and will be used for making ethanol in the near future . logistic study of procuring , transporting and storing these materials are underway . a constant source of lignocellulosic feed stock is mushroom spent straw ( mss ) from a mushroom plant ( obodi et . al , 2003 ). in 2005 , the world market for mushroom is $ 41 billion ( shu - ting chang , 2006 ). over 4 - 5 million tons of edible mushrooms are produced worldwide using different substrates . harvest period and substrate compositions on different days during mushroom growth are given in fig1 a and 1b . traditionally , oyster mushrooms are cultivated using sterile rice straw packed in a polythene bag with spawns distributed at regular intervals followed by storing the bag in humid environment for a period of 50 days . about 100 g mushroom ( dwb ) can be harvested at three intervals starting from 1 kg rice straw ( dwb ). in order to conserve both lignin and hemicellulose for getting maximum sugars , microbial conditioning was stopped after 35 days . further , mushroom spent straw ( mss ) was extracted with water to remove enzymes / proteins , organic acids , soluble lignin and oligosaccharides . the higher molecular weight components (& gt ; 10 kda ) namely enzymes , soluble lignin polymer and oligosaccharides were removed using membrane filtration as retentate . while the low molecular weight components like lignin degradation products , organic acids , lower molecular weight enzymes / proteins were obtained as filtrate . the detailed mass balance for the above washing protocol is given in fig2 . there are several advantages in using the mushroom spent straw ( mss ) because it is available in plenty in pre - processed form . in other words , the microbes degrade the lignin using various combinations of enzymes leaving behind the hemicellulose and cellulose which could be easily accessed during enzymatic hydrolysis . potential for reducing severity of thermo chemical biomass pretreatment was demonstrated for microbial pretreated biomass ( keller et . al ., 2003 ). a substantial amount of composition change ( fig1 a and 1b ) was noticed during the process ( taniguchi et . al ., 2005 ). a combination of key enzymes like , lacases , manganese peroxidase , lignin peroxidase and phenol oxidase were secreted by white rot fungus ( table 1 ) and synergistically degrade lignin ( martinez et . al ., 2005 ). since lignin is the most recalcitrant molecule during the enzymatic hydrolysis , removing them prior to hydrolysis would be expected to provide higher sugar yields . as expected , when mushroom spent straw ( mss ) was hydrolyzed , we could obtain a sugar conversion of up to 40 % using 15 fpu of commercial cellulase enzymes , compared to just 20 % for untreated rice straw . hence , a second pretreatment is required for getting even higher sugar yields . in one of our previous studies , pretreatment of rice straw using afex ( 2 : 1 ammonia to biomass , 40 % moisture , 90 ° c .) could achieve maximum 70 - 80 % glucan conversion ( gollapali et al ., 2002 ) using a much higher cellulase loading of 75 fpu / gm glucan . in the new approach , we used mushroom spent straw ( mss ) using pleurotus ostreatus strain after washing in water , followed by afex pretreatment ( at fixed temperature of 90 ° c ., varying biomass to ammonia ratios from 1 : 1 to 1 : 2 and varying moistures ) to achieve close to 80 % glucan conversion using just 15 fpu / g glucan loading of cellulase enzyme in under 72 hours ( fig3 a and 3b ). picture showing both rice straw ( rs ) and mushroom spent straw ( mss ), before and after afex treatment is given in fig4 a to 4 d . less severe afex conditions are required to achieve the same conversions for mushroom spent straw ( mss ). increasing enzyme loading or increasing the reaction time , further improves the glucan / xylan conversions ( results not shown ). by the present invention , the waste material could be completely hydrolyzed by pretreatment using afex and further can be fermented to ethanol . a preliminary analysis on various metabolites generated during the solid state fermentation process was performed by analyzing the wash stream using hplc . compared to untreated rice straw , mss treated rice straw showed around 10 different organic acids ( results not shown ). in addition , we can extract some enzymes as reported before ( baldrian and gabriel , 2002 ; christian et . al ., 2005 ) which have a wide range of applications in degrading xenobiotic compounds and aromatic compounds in dye industry . at the end of the process , about 10 - 14 % of silica was left behind which could be a valuable inorganic resource for some specific applications . soluble lignin which was extracted along with protein and polypeptides are also considered as one of the important byproducts in the whole process . the enzymes and organic acids removable from the process are shown in table 1 . in addition , enzyme combination studies can be used in order to improve the xylan conversion . higher solid loading during enzymatic hydrolysis can be used followed by fermenting the sugars to ethanol using yeast strain . 1 . baldrian , p , gabriel , j . 2002 . variability of laccase activity in the white - rot basidomycete pleurotus ostreatus . folia microbiol . 47 : 385 - 390 . 2 . cohen r , persky , l , hadar , y . 2002 . biotechnological applications and potential of wood - degrading mushrooms of the genus pleurotus . appl . microbiol . biotechnol . 58 : 582 - 594 . 4 . christian v , shrivastava r , shukla d , modi h a and vyas b r m . 2005 . degradation of xenobiotic compounds by lignin - 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1966 . 26 . williams b c , mcmullan j t , mccahey s . 2001 . an initial assessment of spent mushroom compost as a potential energy feedstock . bioresour . technol . 79 : 227 - 30 . 27 . world mushroom industry production report prepared by usda economics and statistics system & lt ; www . : http :// usda . mannlib . cornell . edu / data - sets / specialty / 94003 /& gt ;. 28 . zhang r , li x , fadel j g . 2002 . oyster mushroom cultivation with rice and wheat straw . bioresour technol . 82 : 277 - 84 . while the present invention is described herein with reference to illustrated embodiments , it should be understood that the invention is not limited hereto . those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof . therefore , the present invention is limited only by the claims attached herein .