Sugarcane cultivar selection for ethanol production using dilute acid pretreatment, enzymatic hydrolysis and fermentation

Benjamin, Yuda L. (2014-04)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: The development of ―energycane‖ varieties of sugarcane for ethanol production is underway, targeting the use of both sugar juice (first generation ethanol) and bagasse (second generation ethanol). Nevertheless, identification of the preferred varieties represents the biggest challenge to the development of energycane due to large number of samples produced during breeding. In the present study, dilute acid pretreatment, enzymatic hydrolysis and fermentation processes were used to evaluate the processability of bagasse (fibrous residue generated after juice sugar extraction) from different varieties of sugarcane to select preferred varieties with the properties of improving combined ethanol yield (ethanol from juice and bagasse) per hectare. The impact of variety selection on combined ethanol yield (ethanol from juice and bagasse) per hectare was also assessed. In the first part of this study, 115 varieties of sugarcane originated from classical breeding and precision breeding (genetic engineering) were screened based on agronomic data and experimental data from biochemical processes (dilute acid pretreatment and enzymatic hydrolysis) applied to the bagasse fraction of each variety. The results showed wide variations in the chemical composition of bagasse between the varieties. Structural carbohydrates and lignin content ranged from 66.6 to 77.6% dry matter (DM) and 14.4 to 23.1% DM, respectively. The majority of precision breeding varieties showed higher arabinoxylan, lower lignin and lower ash content than most of classical breeding varieties. Combined sugar yield from the bagasse after pretreatment and enzymatic hydrolysis also varied significantly among the varieties. Up to 27.9 g/100g (dry bagasse) difference in combined sugar yield was observed. Combined sugar yield was inversely correlated with lignin as well as ash content, but it correlated positively with structural carbohydrates content. Total potential ethanol yields per hectare, calculated based on cane yield, soluble and non-soluble sugar content also differed significantly among the varieties (8,602−18,244 L/ha). Potential ethanol from bagasse contributed approximately one third of the total potential ethanol yield. Interestingly, some of the varieties had combined properties of high potential ethanol yield per hectare and improved bagasse convertibility. Thus, six varieties (3 from each breeding technology) were selected as preferred varieties for further investigation. To enhance sugar yield from bagasse, optimisation of pretreatment was conducted on the selected varieties. Industrial bagasse was included for comparison purposes. The pretreatment optimisation was based on maximising combined sugar yield from the combined pretreatment-hydrolysis process. A central composite design (CCD) was applied to investigate the effects of temperature, acid concentration and residence time on the responses and was later used to determine the maximum combined sugar yield. Pretreatment optimisation was conducted at gram scale (22.9 ml reactor) and at bench scale (1000 ml reactor). Significant differences in sugar yields (xylose, glucose, and combined sugar) between the varieties were observed. The combined sugar yields from the best performing varieties and industrial bagasse at optimal pretreatment-hydrolysis conditions differed by up to 34.1% and 33% at gram and bench scale, respectively. A high ratio of carbohydrates to lignin and low ash contents increased the release of sugar from the substrates. At mild pretreatment conditions, the differences in bioconversion efficiency between varieties were greater than at severe conditions. This observation suggests that under less severe conditions the conversion efficiency was largely determined by the properties of the biomass. Furthermore, it was demonstrated that the pretreatment conditions with temperature ranged from 184 to 200 °C and varying residence time to provide a severity factor between 3.51 and 3.96 was observed to be the area in common where 95% of maximum combined sugar yield could be obtained. Simultaneous Saccharification and Fermentation (SSF) was performed on the unwashed pressed-slurry from bagasse pretreatment at conditions for maximum combined sugar yield at bench scale. Batch and fed-batch SSF feeding strategy at different solid loadings and enzyme dosages were used aiming to reach an ethanol concentration of at least 40 g/L. The results revealed significant improvement in overall ethanol yield after SSF for the selected varieties (84.5–85.6%) compared to industrial bagasse (74.8%). The maximum ethanol concentration from the best performing varieties was 48.6−51.3 g/l and for poor performing varieties was 37.1−38.3 g/l. Ethanol concentration in the fermentation broth was inversely correlated with lignin content and the ratio of xylose to arabinose, but it showed positive correlation with glucose yield from pretreatment-enzymatic hydrolysis. The overall assessment of the varieties showed greater improvement in combined ethanol yields per hectare (71.1–90.7%) for the best performing varieties with respect to industrial sugarcane. The performance in terms of ethanol yields of selected varieties from a number harvest years was evaluated. The results showed considerable variations in ethanol yields across harvests. The results showed that the best variety in terms combined ethanol yield was not maintained across harvests. The differences in ethanol yields were greater among the varieties than across the harvests. Prolonged severe drought significantly affected the ethnol yields of all varieties represented by lower and intermediate lignin content for cane yield compared to that which had highest lignin content. However, carbohydrates content in the bagasse and sugar yield/recovery between the harvest years did not change for the most of the varieties. In summary, the present study provides evidence of the impact of cultivar selection and pretreatment optimisation in increasing conversion efficiency of bagasse. The results demonstrate that varieties with lower lignin and ash content, as well as highly substituted xylan resulted in higher sugar and ethanol yields. These results suggest that lower process requirements can be achieved without adversely affecting juice ethanol and cane yield per hectare. Nonetheless, an attempt to reduce lignin content in the bagasse, to reduce processing requirements for ethanol production, can also target the improvement of crop tolerance toward severe drought conditions.

AFRIKAANSE OPSOMMING: Die ontwikkeling van ―energie-riet‖ rasse vir etanol produksie is goed op dreef, waar beide die sap (eerste generasie etanol) en die bagasse (tweede generasie etanol) geteiken word. Die groot aantal monsters wat tydens teling geproduseer word, bied egter die grootste uitdaging vir die identifisering van nuwe rasse ten einde energie-riet te ontwikkel. In die huidige studie is verdunde suurvoorbehandeling, ensiematiese hidrolise en fermentasie-prosesse gebruik om die verwerkbaarheid van bagasse (veselagtige residu gegenereer na sap suiker ekstraksie) van verskillende suikerrietrasse te evalueer om nuwe variëteite te selekteer wat eienskappe van verbeterde gekombineerde etanolopbrengs (etanol van sap en bagasse) per hektaar toon. Die impak van variëteit-seleksie op gekombineerde etanol opbrengs (etanol van sap en bagasse) per hektaar is ook beoordeel. In die eerste deel van hierdie studie het uit ‗n siftingsproses van 115 suikerriet rasse bestaan wat deur klassieke en presisie (geneties gemodifiseerde) teling gegenereer is. Die sifting was op agronomiese data gebaseer, asook op data van verdunde suur voorafbehandeling en ensimatiese hidrolise eksperimente wat op die bagasse fraksie van elke ras uitgevoer is. Die resultate het op groot variasie in die chemiese samestelling van die bagasse van verskillende rasse gedui. Die strukturele koolhidrate het tussen 66.6 en 77.6% droë massa (DM) gewissel, terwyl die lignien inhoud ‗n variasie van 14.4 en 23.1% DM getoon het. Verder het meeste van die presisie-teling variëteite ‗n hoër arabinoxilaan, maar ‗n laer lignien en as-inhoud as meeste van die klassieke teling rasse gehad. Die gekombineerde suikeropbrengs (GSO) van die bagasse na voorafbehandeling en ensimatiese hidrolise het ook beduidend tussen rasse gewissel, waar ‗n verskil van tot 27.9 g/100g (droë bagasse) waargeneem is. Daar was ‗n omgekeerde korrelasie tussen die gekombineerde suikeropbrengs en die lignien en as-inhoud gewees, maar die opbrengs het ‗n sterk positiewe korrelasie met die strukturele koolhidrate getoon. Die totale potensiële etanol opbrengs per hektaar wat vanaf die suikerriet se oplosbare en nie-oplosbare suikerinhoud bereken is, het ook beduidend tussen rasse verskil (8,602−18,244 L/ha), waar die potensiële etanol opbrengs van die bagasse gedeelte ongeveer een derde van die totale potensiële etanol opbrengs beslaan het. Interessante bevindinge het op sommige rasse met gekombineerde eienskappe van hoë potensiële opbrengs per hektaar asook ‗n hoë omskakelingsvermoë gedui. Derhalwe is ses variëteite (drie van elke telingstegnologie) as voorkeurvariëteite vir verdere studie gekies. Om die etanol opbrengs vanaf die bagasse te verbeter was voorafbehandeling van die voorkeurvariëteite geoptimeer, en waar industriële bagasse vir vergelykingsdoeleindes ingesluit was. Vir die optimering was dit ten doel gestel om die gekombineerde suikeropbrengs van die gekombineerde voorafbehandeling-hidrolise proses te maksimeer. ‗n Sentrale saamgestelde ontwerp (SSO) is gebruik om die effek van temperatuur, suurkonsentrasie en residensietyd op die responsveranderlikes vas te stel wat uiteindelik gebruik is om die maksimum gekombineerde suikeropbrengs te bepaal. Die optimering van die voorafbehandeling is op gram-skaal in ‗n 22.9 ml reaktor, asook op bank-skaal in ‗n 1000 ml reaktor uitgevoer. Beduidende verskille in die suikeropbrengs (xilose, glukose en gekombineerde suiker) is tussen die voorkeurrasse waargeneem. Tussen die rasse wat die beste gevaar het, asook die industriële bagasse, het die gekombineerde suikeropbrengs by optimale voorafbehandeling-hidrolise toestande onderskeidelik met tot 34.1% en 33% op gram-skaal en bank-skaal gevarieer. ‗n Hoë verhouding van koolhidrate tot lignien, asook ‗n lae as-inhoud het tot ‗n toename in die vrystelling van suiker uit die substraat gelei. By matige voorafbehandelingstoestande was die verskille in omskakelingseffektiwiteit tussen rasse groter as onder hewige toestande, wat daarop gedui het dat omskakelingseffektiwiteit grotendeels deur die eienskappe van die biomassa bepaal is. Verder is daar ook gedemonstreer dat die voorbehandelingsomstandighede met temperatuur tussen 184 en 200ºC en verandering van die residensietyd om 'n hewigheidsfaktor van tussen 3.51 en 3.96 te verskaf, 'n gemeenskaplike area gelewer het waar 95% van maksimum gekombineer suiker opbrengs (GSO) verkry kon word. Gelyktydige versuikering en fermentasie (GVF) is na voorafbehandeling op ongewaste, gepersde bagasse substraat by toestande vir die maksimum gekombineerde suikeropbrengs op bank-skaal uitgevoer. Bondel en voerbondel SSF voerstrategie by verskillende vaste ladings en ensiemdoserings is gebruik om 'n etanol konsentrasie van ten minste 40 g/L te bereik. Ná GVF was die algehele etanol opbrengs vir die voorkeurvariëteite (84.5–85.6%) beduidend beter relatief tot die industriële bagasse (74.8%). Die maksimum etanol opbrengs na SSF van die rasse met die beste prestasie was 48.6-51.3 g/L en 37.1-38.3 g/L vir rasse wat swak presteer het. Die etanol konsentrasie in die fermentasiesop was omgekeerd met lignien en die verhouding van xilose tot arabinose gekorreleer, maar was duidelik positief met die glukose opbrengs vanaf voorafbehandeling-hidrolise gekorreleer. ‗n Algemene assessering het op ‗n duidelike verbetering van die voorkeurvariëteite in terme van gekombineerde etanol opbrengs per hektaar gedui (71.1–90.7%), relatief tot die industriële suikerriet. Die prestasie in terme van etanol opbrengs van geselekteerde variëteite is oor 'n reeks oesjare ge-evalueer. Die resultate het aansienlike variasies in etanol opbrengs oor oesjare getoon. Die resultate het gewys dat die beste variëteite in terme van gekombineerde etanol opbrengs nie volhou is oor oeste nie. Die verskille in etanol opbrengste tussen variëteite was groter as die verskille oor oesjare. Verlengde ernstige droogte het die etanol opbrengs van alle variëteite met laer en intermediere lignien inhoud vir rietopbrengs aansienlik beinvloed, in vergelyking met dié wat die hoogste lignien inhoud gehad het. Die koolhidraatinhoud in die bagasse en suiker opbrengs/lewering tussen die oesjare het vir die meeste variëteite egter nie gewissel nie. Ter opsomming, die huidige studie verskaf bewyse van die impak van kultivarseleksie en voorbehandelings optimisering op die verhoging van die omskakelings-doeltreffendheid van bagasse. Die resultate wys dat variëteite met laer lignien- en asinhoud, en hoogs-gesubstitueerde xilaan hoër suiker- en etanol opbrengs gelewer het. Hierdie resultate stel voor dat verminderde voorbehandelingsvereistes bereik kan word sonder om die sap etanol en rietopbrengs per hektar te benadeel. Nieteenstaande, 'n poging om die lignien inhoud van die bagasse te verminder om die verwerkingsvereistes vir etanolproduksie te verminder, kan ook die verbetering van gewas-toleransie tov ernstige droogte-toestande teiken.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/86525
This item appears in the following collections: