|HAO WAKATIMWITH US009856500B2 OULULAH M (12 ) United States Patent ( 10 ) Patent No. : US 9 , 856 ,500 B2 Adhikari et al. (45 ) Date of Patent: Jan . 2 , 2018 ( 54 ) METHOD OF CONSOLIDATED ( 56 ) References Cited BIOPROCESSING OF LIGNOCELLULOSIC BIOMASS FOR PRODUCTION OF L - LACTIC U . S . PATENT DOCUMENTS ACID 2005 /0106694 A1 * 5 /2005 Green .. .. .. C12R 1 /07 435 / 146 ( 71) Applicant : Council of Scientific and Industrial Research , New Delhi ( IN ) FOREIGN PATENT DOCUMENTS (72 ) Inventors : Dilip Kumar Adhikari, Mohkampur AU WO 2007140521 A1 * 12/ 2007 .. A61K 8 /97 ( IN ) ; Jayati Trivedi, Mohkampur ( IN ) ; WO WO 2012 / 071392 A2 * 5 / 2012 C12N 1 /21 Deepti Agrawal, Mohkampur ( IN ) WO WO - 2014 /013509 1 / 2014 (73 ) Assignee : Council of Scientific and Industrial OTHER PUBLICATIONS Research , New Delhi ( IN ) Taxonomy Paenibacillus macerans (Bacillus macerans ) (Species Paema Taxon Identifier http : / /www .uniprot .org / taxonomy/ 44252 ( * ) Notice : Subject to any disclaimer, the term of this printed Sep . 29 , 2016 . * patent is extended or adjusted under 35 Nakamura et al . 1988 . Taxonomic Study of Bacillus coagulans Hammer 1915 with a Proposal for Bacillus smithii sp . nov . Inter U . S . C . 154 ( b ) by 201 days . national Journal of Systematic Bacteriology , vol . 38 , pp . 63 - 73. * Ryckeboer et al . 2003 . Microbiological aspects of biowaste during (21 ) Appl . No. : 14 /415 , 652 composting in a monitored compost bin . Journal of Applied Micro biology , vol. 94 , 127 - 137 . * (22 ) PCT Filed : Jul . 17, 2013 Partanan et al 2010 . Bacterial diversity at different stages of the composting process . BMC Microbiology vol. 10 , pp . 94 - 104 ( 1 - 11) . * ( 86 ) PCT No. : PCT/ IN2013 /000443 Sigma- Aldrich , 2 pages . http :/ /www . sigmaaldrich . com / catalog $ 371 (c ) ( 1) , search ? term = carboxymethylcellulose & interface = All; Printed Mar . 15 , 2017 . * ( 2 ) Date : Jan . 19 , 2015 Kadam et al . 2003 . Availability of corn stover as a sustainable feedstock for bioethanol production . Bioresource Technology , vol . (87 ) PCT Pub . No .: WO2014 /013509 88 , pp . 17 - 25 . * “ International Application No . PCT/ IN2013 /000443 , International PCT Pub . Date : Jan . 23 , 2014 Search Report dated Nov . 28 , 2013” , (dated Nov . 28 , 2013 ), 4 pgs . (65 ) Prior Publication Data * cited by examiner US 2015/ 0197777 A1 Jul. 16 , 2015 Primary Examiner — Pablo S Whaley (30 ) Foreign Application Priority Data Assistant Examiner — Kailash C Srivastava ( 74 ) Attorney, Agent, or Firm — Schwegman Lundberg & Jul . 19 , 2012 (IN ) . .. 2237 /DEL / 2012 Woessner, P . A . (51 ) Int. Ci. (57 ) ABSTRACT C12P 7 /56 ( 2006 .01 ) The present invention pertains to a method for consolidated CO2F 3130 ( 2006 . 01 ) bio processing of lignocellulosic biomass to L -Lactic acid . C12N 1 / 20 ( 2006 .01 ) Particularly , the present invention relates to the production C12R 1 /01 ( 2006 .01 ) of L - Lactic Acid from low cost non edible feedstock ligno B09C 1 / 10 ( 2006 .01 ) cellulosic biomass . More particularly the present invention (52 ) U . S . Cl. relates to the process for one step production of L - Lactic CPC .. C12P 7 /56 ( 2013. 01 ) ; C12N 1/ 20 Acid from lignocellulosic biomass using thermophilic bac (2013 .01 ) ; C12R 1/ 01 ( 2013 .01 ) ; C12P teria Paenibacillus macerans IIPSP3 (MTCC 5569 ) , which 2203/ 00 ( 2013 . 01 ) is not only capable of hydrolyzing cellulose to glucose but (58 ) Field of Classification Search also further fermenting it to L -Lactic Acid under aerobic CPC .. .. .. C12N 1 /20 ; C12P 2203 /00 , C12P 7 /56 ; conditions, without any growth inhibition in presence of C12R 1 /01 ; Y10S 435 / 911; B01J lignin . The present invention provides a process which has 2220 /4825 less chances of contamination , as the fermentation is carried USPC . .. .. 435 /254 . 1 , 262. 5 , 911 , 29 , 32 , 34 , 41, out at higher temperatures and is economically attractive , as 435 / 139 preferably no external enzyme loadings are required . IPC .. .. B09C 1 / 10 ; CO2F 3 /30 ; C12P 7 /56 See application file for complete search history . 11 Claims, 2 Drawing Sheets U . S . Patent Jan . 2 , 2018 Sheet 1 of 2 US 9 ,856 ,500 B2 (A ) wwwwwwwwwwwww - - - - - - WWW Y * * * * * * * * * * * * * w (8 ) 3 20 S : Fig . 1 i in Niiw Lacticamountof * 2401 oforma important X48 : ius * 77 * : a S 12 20 illaAmount of Glucose in Fermentation Media ( g ) Fig . 2 U . S . Patent Jan . 2 , 2018 Sheet 2 of 2 US 9 ,856 ,500 B2 25. 0 225 * . * . * , * , 4 , 4 , 4 , 4 , 4 , 4 , 4 , , 4 , 4 , 4 , 4 , 4 , 4 , 4 , 4 , 4 , 4 , 4 , 4 , 4 , * . * . * . * . * . * * * . * . * . * . * . * . * . * . * . * . * . * . * . * . * . * . * . * , * . * * * . * . * . * . * . * . * . * . * . * . * . * . * . * . * . * . * . * . 20 .0 37 . 5 . .. - - - - - - - - - - - - - - - - 15 . 0 (runout) mai Concentration mult . * * * * * * * * * ** * * * * .. .. .. 4 9 12 16 20 24 Fermentation Period (Hours ) Cellobiose utilization LaCucacd Formation Fig . 3 ". - - - LacticAcid qudu)tonulos - - entre - 0 5 10 15 20 25 30 Fermentation Perod Hours ) Fig . 4 US 9 , 856 , 500 B2 METHOD OF CONSOLIDATED stock resources such as corn , whey, potatoes , cane sugar, BIOPROCESSING OF LIGNOCELLULOSIC molasses of beet sugar , xylose stream generated from ligno BIOMASS FOR PRODUCTION OF L - LACTIC cellulosic biomass etc . ACID U . S . Pat. No . 4 , 963 ,486 issued to Hang , on Oct . 16 , 1990 5 claims the production of L ( + ) lactic acid from Rhizopus PRIORITY CLAIM TO RELATED oryzae as a single step fermentation process from renewable APPLICATIONS biomass namely corn , cassava , rice , oat, wheat, barley and sorghum representing starchy biomass . The patent claims that the fungus mentioned in the invention is capable of This application is a U . S . national stage application filed1 10 supplying all the required enzymes for both saccharification under 35 U . S . C . $ 371 from International Application Serial of gelatinised starch and fermentation to L ( + ) - lactic acid . No . PCT/ IN2013 /000443 , which was filed Jul. 17 , 2013 , and Hang also discloses the production of 350 g of L ( + ) - lactic published as WO 2014 /013509 on Jan . 23, 2014 , and which acid from one kg crushed corn . claims priority to India Application No . 2237 /DEL / 2012 , Similarly Tsai et al in their U . S . Pat . No . 5 ,464 , 760 ( 7 filed Jul. 19 , 2012 , which applications and publication are 15 Nov . 1995 ) claim the production of lactic acid using a incorporated by reference as if reproduced herein and made consortium of Lactobacillus strains from Starch under SSF a part hereof in their entirety , and the benefit of priority of conditions in combination with a - amylase enzyme and each of which is claimed herein . thereafter its recovery in pure form from Sodium lactate using various methods like electro - dialysis . The invention of FIELD OF THE INVENTION 20 U . S . Pat. No . 5 , 464 , 760 involves the bioconversion of industrial food waste , such as potato waste , corn , rice , The present invention pertains to a method for consoli cheese whey, cane sugars , beet sugars or the like , containing dated bio processing of lignocellulosic biomass to L - Lactic starch to lactic acid suitable for conversion to photodegrad acid . Particularly , the present invention relates to the pro - able or biodegradable plastics . duction of L - Lactic Acid from low cost non edible feedstock 25 The European Patent Application filed by Shimadzu Cor lignocellulosic biomass . More particularly the present poration (EP0770684A2 ) with a priority date of 27 Oct. invention relates to the process for one step production of 1995 , claims the production of L - Lactic Acid with a purity L - Lactic Acid from lignocellulosic biomass using thermo - of more than 70 % to as high as 95 % by Bacillus species in philic bacteria Paenibacillus macerans IIPSP3 (MTCC particular under anaerobic conditions mainly from glucose , 5569) , having been deposited under accession number 30 sucrose, maltose , fructose , mannitol , lactose and Starch . MTCC 5569 on Jun . 28 , 2010 under the Budapest Treaty at Numerous papers and patents have reported the produc tion of L ( + ) - lactic acid through simultaneous saccharifica Microbial Type Culture Collection & Gene Bank , Institute tion and fermentation (SSF ) route . However one of the of Microbial Technology , Sector 39 - A , Chandigarh 160 036 major disadvantage quoted by Hofvendahl and Hahn -Hag India , which is not only capable of hydrolysing cellulose to 35 erdal (Enzyme Microb . Technol. , 2000 ; 26 : 87 - 107) with the glucose but also further fermenting it to L -Lactic Acid under SSF process is the difference in the optimal conditions for aerobic conditions, without any growth inhibition in pres enzymatic hydrolysis ( pH < 5 . 0 and temperature : 50° C . ) and ence of lignin . lactic acid fermentation (pH - 5 . 0 - 7 . 0 and temperature being It is further appreciated that the process has less chances 37 - 43° C .) . of contamination , as the fermentation is carried out at higher 40 Shin -ichiro Abe and Motoyoshi Takagi have shown the temperatures and is economically attractive , as preferably no production of lactic acid using a combination of external enzyme loadings are required . Trichoderma reesei as a source of cellulase enzyme for saccharification and Lactobacillus delbrueckii as the lactic BACKGROUND OF THE INVENTION acid producing microorganism . At the end of 120 hours 52 . 5 45 g / L of lactic acid was produced with only 6 . 2 g / L of reducing Lactic Acid or 2 -Hydroxypropionic