Optimisation of a simultaneous saccharification and fermentation process for use with steam pretreated sweet sorghum bagasse

Dreyer, Casper Becker (2013-12)

Thesis (MScEng)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Global warming and greenhouse gas (GHG) emissions are serious issues of our time. One of our greatest sources of pollution originates from the combustion of fossil fuels where the largest contributor, on a global scale, is the transport sector. Renewable sources of liquid fuels have been investigated with bioethanol being the most widely used worldwide. Production of first generation bioethanol from food crops raised concerns over food supplies being channelled for biofuel production leading to possible increases in food prices. Second generation or lignocellulosic bioethanol from the residual plant material does not compete with food crops while dedicated energy crops could be cultivated on land that is not suited for food production by agriculture. Of the various crops investigated, sweet sorghum appears to be a viable candidate for lignocellulosic ethanol production since this crop can be cultivated in a range of soil conditions whilst low water requirements make it ideal for arid regions. The sweet juice extracted from the sweet sorghum stem can be utilised for first generation biofuel production in conjunction with the bagasse in a second generation plant. In order to ensure economic feasibility for industrial-scale lignocellulosic ethanol production, it is critical that the process as a whole be optimised to minimize expenditure while maximising yields. In the current study a fed-batch simultaneous saccharification and fermentation process was developed for use with steam-pretreated sweet sorghum bagasse at high solid loadings. Two separate studies were conducted in parallel with the first focusing on selecting the preferred sweet sorghum varieties and optimising the steam pretreatment conditions while the second focussed on optimising the enzyme cocktail required for hydrolysis, which occurs simultaneously with fermentation. The developed SSF process was investigated by utilising the preferred pretreatment conditions and enzyme cocktails and further optimised in terms of solid loadings and feeding to achieved 40 g/L of ethanol. Preliminary fermentation runs were performed to identify the mains issues with the SSF process. For these runs sweet sorghum bagasse of variety MSJH13, pretreated dry (7.3% moisture) at 200 °C for 5 minutes, with an enzyme dosage of 0.167 ml Cellic CTec2/g dry WIS, was utilised. Investigation of the three process options of whole slurry, pressed WIS and washed WIS resulted in low ethanol concentrations and accumulation of glucose. The performance of three recombinant strains of Saccharomyces cerevisiae (D5A⁺, D5A⁺ᴴ and TMB3400) and one wild-type industrial strain (MH1000) were investigated under SSF conditions. Strain MH1000 delivered the highest ethanol concentration of 34.5 g/L from a cumulative solids loading of 20% using pressed WIS compared to 32.2 g/L for strain D5A⁺ᴴ under the same conditions. At a solid loading of 20%, yeast growth and fermentation inhibition coupled with glucose accumulation was observed, due to inhibitor concentrations reaching critical levels. Strain MH1000 showed increased tolerance to the inhibitor concentrations and was only inhibited after 94h compared to 76h for strain D5A⁺ᴴ. To optimise the SSF process the preferred sweet sorghum varieties SS27 and AP6 were pretreated with water-impregnation at 205 °C for 5 minutes while variety SS27 was also pretreated after impregnation with 3% SO2 at 185 °C for 8 minutes. Two enzyme cocktails of 0.15 ml Cellic CTec2/g dry WIS and containing either 0.32 ml Cellic HTec2/g dry WIS or 0.017 ml Cellic HTec2/g dry WIS (referred to as Cocktail 1 and Cocktail 2) were also investigated. At cumulative solid loadings of 20% in fed-batch SSF using the water-soaked material, yeast inhibition and glucose accumulation was observed, irrespective of the enzyme cocktail utilised. The 18-fold increase in the Cellic HTec2 concentration from Cocktail 1 did not significantly increase the ethanol productivity or ethanol concentration obtained, compared to Cocktail 2 which resulted in 43.6 g/L of ethanol. By reducing the cumulative solids loading to 16% and 13%, glucose accumulation was reduced and avoided for the respective loadings while the maximum ethanol concentration only decreased to 41.4 g/L and 38.9 g/L respectively. An ethanol yield of 82% of the theoretical maximum, based on the glucose added to the fermentation broth, was calculated for fermentation with the cumulative solid loading of 13% using the water-soaked material form variety SS27. In literature a similar yield of 75% has been reported for a solids loading of 16%, but washing of the pretreated material prior to SSF was required. Using pretreated material from variety SS27, impregnated with 3% SO2 as catalyst during steam pretreatment, at a solids loading of 13% and Cocktail 2 resulted in a maximum ethanol concentration of 36.8 g/L and a productivity of 0.298 g/L.h with a yield of 79% of the theoretical maximum (based on glucose). The differences, compared to the water-soaked only pretreatment, were not statistically significant. Similarly sweet sorghum varieties AP6, pretreated after water soaking, under SSF conditions also exhibited no significant differences compared to variety SS27 with regards to the maximum ethanol concentration (35.8 g/L) and productivity (0.289 g/L.h) obtained and a yield of 75% of the theoretical maximum (based on glucose) was calculated.

AFRIKAANSE OPSOMMING: In die moderne era word groot klem gelê op die effekte van aardverwarming weens kweekhuisgasse. Op ʼn wêreldwye skaal is die grootste bron van kweekhuisgasse afkomstig vanaf die verbranding van fossielbrandstowwe soos gebruik deur die vervoerindustrie. Van die alternatiewe brandstowwe wat ondersoek word, blyk bio-etanol die belowendste te wees. Eerstegenerasie bio-etanol afkomstig vanaf voedselbronne soos mielies het gelei tot etiese kwessies vanweë die wêreld voedseltekort. Tweedegenerasie of lignosellulose etanol maak gebruik van residuele plant materiale of energiegewasse was verbou kan word op grond wat ongeskik is vir boerdery bedrywighede en hou dus geen bedreig vir voedselverskaffing in nie. In Suid-Afrika blyk soetsorghum ʼn ideale energiegewas te wees vir die produksie van tweedegenerasie bio-etanol aangesien hierdie gewas verbou kan word ‘n wye reeks grond kondisies. Die sap afkomstig vanaf soetsorghum kan gebruik word vir etanol produksie in ‘n eerstegenerasie proses terwyl die oorblywende plantmateriaal gebruik kan word in die tweedegenerasie proses. Om die ekonomiese lewensvatbaarheid van die proses te verseker, moet proses optimering ondersoek word. Dit was genoodsaak om die gelyktydige hidroliese en fermentasie (GHF of SSF in Engels) van stoom behandelde soetsorghum bagasse in ʼn semi-enkellading proses te ondersoek. Studies wat in parallel uitgevoer was, het gefokus op die seleksie van soetsorghum kultivars en optimering van die kondisies vir stoom behandeling en die bepaling van die ensiempreparaat vir hidroliese. Die optimeerde stoom behandelings kondisies en ensiempreparaat was ondersoek tydens SSF tesame met verder optimering van die SSF proses om ten einde ’n etanol konsentrasie van 40 g/L te verkry. Vir ontwikkeling van die SSF proses was monsters van soetsorghum kultivar MSJH13 met ‘n vog-inhoud van 7.3%, behandel teen 200 °C vir 5 minute en ʼn ensiem lading van 0.167 ml Cellic CTec2/gram droë materiaal was gebruik. Ondersoek van die drie moontlike proses opsies van slurry, pressed WIS en washed WIS was onbevredigend vanweë die lae etanol konsentrasies en akkumulasie van glukose. Drie rekombinante gis rasse van Saccharomyces cerevisiae (D5A⁺, D5A⁺ᴴ en TMB3400) en een industriële ras (MH1000) was ondersoek. Ras MH1000 het die hoogste etanol konsentrasie gelewer (34.5 g/L) vanaf ‘n materiaallading van 20% en ook die stertste weerstand teen inhibitore gebied. ‘n Akkumulasie van glukose weens inhibisie van die gis was waargeneem vir ‘n materiaallading van 20%, vanweë die konsentrasie van inhibitore in die proses. Vir optimering van die SSF proses was monsters van soetsorghum kultivars SS27 en AP6 behandel na water-benatting teen 205 °C vir 5 minute en ʼn tweede monster van SS27 was ook behandel met 3% SO2 teen 185 °C vir 8 minute. Twee ensiemdosisse was ondersoek en beide het 0.15 ml Cellic CTec2/gram droë materiaal bevat met onderskeidelik 0.32 ml Cellic HTec2/gram droë materiaal of 0.017 ml Cellic HTec2/gram droë materiaal (Cocktail 1 en Cocktail 2). Met ‘n materiaallading van 20% was inhibisie van die gis en akkumulasie van glukose ondervind, ongeag die ensiemdosis wat gebruik was. Die hoë ensiemdosis van Cocktail 1 het nie ‘n beduidende verhoging in die etanol konsentrasie veroorsaak nie, in vergelyking met Cocktail 2 wat 43.6 g/L etanol gelewer het. Deur die materiaallading te verlaag tot 13% kon inhibisie van die gis voorkom word en is ‘n etanol konsentrasie van 38.9 g/L verkry teen ‘n etanol opbrengs van 82% van die teoretiese maksimum (gebaseer op die glukose in die fermentasie). ‘n Vergelyking met SO2 behandelde materiaal (teenoor water-benatte materiaal van kultivar SS27) teen ‘n materiaallading van 13% het geen statistiese beduidende verskille opgelewer met betrekking tot die maksimum etanol konsentrasie (36.8 g/L) of produktiwiteit (0.298 g/L.h) nie terwyl ‘n opbrengs van 79% bereken was. Vergelyking met soetsorghum AP6, ook water-benat, in SSF met ‘n materiaallading van 13% het ook geen beduidende verskille opgelewer in terme van maksimum etanol konsentrasie (35.8 g/L) of produktiwiteit nie (0.289 g/L.h).

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