TWI382092B - 提升纖維水解液生產五碳糖醇之酵母菌培養方法 - Google Patents
提升纖維水解液生產五碳糖醇之酵母菌培養方法 Download PDFInfo
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- TWI382092B TWI382092B TW098136801A TW98136801A TWI382092B TW I382092 B TWI382092 B TW I382092B TW 098136801 A TW098136801 A TW 098136801A TW 98136801 A TW98136801 A TW 98136801A TW I382092 B TWI382092 B TW I382092B
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- hydrolyzate
- xylose
- pentitol
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- 238000000034 method Methods 0.000 title claims description 47
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims description 32
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 title claims description 14
- 238000004519 manufacturing process Methods 0.000 title description 16
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- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 claims description 17
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- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 description 1
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- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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Description
本發明係有關於一種提升纖維水解液生產五碳糖醇之酵母菌培養方法,尤指涉及一種以特定條件培養酵母菌,特別係指可提升纖維原料水解液中之木糖轉化為五碳糖醇之產率者。
有鑒於生質酒精被視為一種深具潛力可取代汽油之燃料,因此建立可永續發展之生質酒精生產技術已成為進行生質燃料研發之重要議題。基於木材(Wood)、蔗渣(Bagasse)、稻稈(Rice Straw)、玉米桿(Corn Stover)與麥稈(Wheat Straw)等農林業廢棄物;芒草(Silvergrass)與狼尾草等草本植物;以及紙類廢棄物等纖維生質原料,因具有全球儲存量高、來源多樣性以及不會造成糧食衝突之優點,同時亦已被證實具有減碳及增加能源淨產出之效益,故以這些非糧化生質原料轉化酒精已被視為未來最具有潛力之生質酒精生產技術,尤其,在全球已建立發展低碳社會之共識趨勢下,纖維酒精亦被視為發展低碳運輸之重要技術選項。
一般纖維生質原料主要含有60~80%之纖維素與半纖維素,以及15~25%之木質素,其中纖維素與半纖維素以糖化技術(Saccharification Process)分別轉化為六碳糖(主要為葡萄糖)及五碳糖(主要為木糖)後,即能以生物發酵技術將這些單糖進一步轉化為酒精。另一方面,在纖維轉化酒精過程中,現階段多採用稀酸水解前處理技術(Dilute-acid Hydrolysis Pretreatment),將半纖維素分解為木糖,其操作流程係先將一
定比例之生質原料與水溶液混合後裝填於反應器內,再於高溫高壓反應條件下加入1~2%(w/w)之稀硫酸反應數分鐘,因此所得到之木糖發酵液體會含有較高濃度之硫酸根離子,導致影響酵母菌將木糖轉化成酒精之能力。再者,由於利用稀酸水解前處理會同時產生若干濃度之醋酸(Acetic Acid)、糠醛(Furfural)及羥甲基糠醛(Hydroxymethyl Furfural)等之發酵抑制物,進而影響酵母菌發酵能力。因此現階段前處理所得之木糖水解液,通常會先以過鹼化法移除糠醛及硫酸根離子,藉以降低前處理產生之水解液之抑制性。
由於纖維酒精技術發展尚未成熟,生產成本偏高而不具競爭力,因此以纖維酒精生產製程為基礎,併同開發高價副產品之生產技術,將纖維酒精技術提升為生物精鍊製程,近期已形成未來纖維酒精產業之主要發展趨勢。由於以現階段技術層次將木糖轉化為酒精,易發生製程損失、轉化效率偏低及增加酒精純化之能耗等間題,致使許多國外纖維酒精測試廠並未能使用木糖生產酒精,因此如何有效地將木糖轉化為其他高價副產品,藉以提高纖維酒精製程之附加價值,即為發展生物精鍊產業之重要議題。
木糖醇(Xylitol)、阿拉伯糖醇(Arabitol)及核糖醇(Ribitol)等五碳糖醇(Pentitol)係一種自然界存在之少量稀有糖類,可在蔬果等植物組成中發現,也是哺乳類代謝醣類之中間代謝產物之一,其中阿拉伯糖醇係可作為食品添加劑或甜味劑,核糖醇係合成維他命B2
之重要原料,木糖醇則係近年來最受到高度關注者,主要原因為木糖醇係一種與蔗糖甜度相當但熱量只有蔗糖60%之天然甜味劑,除了對於預防蛀牙有顯著之幫助,
在臨床上更廣泛被用作糖尿病患者食物中蔗糖之替代品。以木糖醇為例,現階段工業量產木糖醇之方法亦利用酸水解前處理產生之木糖水解液為原料,惟當製造木糖醇時乃以高溫高壓條件及鎳金屬催化下加氫還原反應之化學合成法為之。雖然以上述化學合成方式係可生產木糖醇,然而其產率僅約在40~50%之間,同時尚有製造過程繁複並需耗費大量能源等缺點。
有鑑於此,以微生物直接發酵纖維原料水解液生產五碳糖醇之生物轉化方式係目前認為最具優勢及競爭力之替代方法,利用自然界存在並可利用木糖之微生物,以微生物本身之生理代謝反應將木糖直接轉化為五碳糖醇,不僅免除化學法中木糖醇產物可能受重金屬污染之風險,並具有低耗能之優點,因此在全球已有建立低碳經濟及低碳社會之理念下,發展低耗能之五碳糖醇生化製程應有其必要性。
基本上,目前已發表研究資料中可用以同時生產多種五碳糖醇之菌株,乃以基因重組菌株為主(Toivari M.H.,et al.,2007,Metabolic Engineering of Saccharomyces cerevisiae for Conversion of D-Glucose to Xylitol and Other Five-Carbon Sugars and Sugar Alcohols,Applied and Environmental Microbiology,p.5471-5476);惟,Candida菌屬之菌株雖然有相當不錯之木糖醇生產效率,但並無法同時產生阿拉伯糖醇及核糖醇等其他五碳糖醇。故,一般習用者係無法符合使用者於實際使用時之所需。
本發明之主要目的係在於,克服習知技藝所遭遇之上述問
題並提供一種以特定條件培養酵母菌Pichia stipitis
為發酵菌株,俾使其可以提升纖維原料水解液中之木糖轉化為木糖醇、阿拉伯糖醇及核糖醇等多種五碳糖醇之產率者。
本發明之次要目的係在於,提供一種以未經去毒性處理之水解液為原料,其提升五碳糖醇產率之幅度會較經過鹼化法處理之水解液之效果為佳,俾將可省略水解液必須先行去毒化調理之需求,進而能相對降低所需之設備投資成本者。
本發明之另一目的係在於,提供一種培養後之酵母菌除了具有生成木糖醇、阿拉伯糖醇及核糖醇等多種五碳糖醇之能力外,尚可同時將木糖轉化為酒精,後續可藉由適當之酒精純化及濃縮技術,併同提供可作為車用燃料之纖維酒精使用者。
為達以上之目的,本發明係一種提升纖維水解液生產五碳糖醇之酵母菌培養方法,係以特定條件培養酵母菌Pichia stipitis
為發酵菌株,應用於未經去毒性(Detoxification)處理或經過鹼化法(Overliming)處理之纖維原料水解液中,俾利於提升該纖維原料水解液中之木糖轉化為五碳糖醇之產率達3~6倍;於其中,該酵母菌之培養條件係添加介於2.0~4.0克/公升(g/L)範圍之醋酸於培養液中,且該培養液係可以葡萄糖、木糖或木糖水解液為碳源培養。
本發明係一種提升纖維水解液生產五碳糖醇之酵母菌培養方法,係以特定條件培養酵母菌Pichia stipitis
為發酵菌株,應用於未經去毒性(Detoxification)處理或經過鹼化法(Overliming)處理之纖維原料水解液中,可提升該纖維原料
水解液中之木糖轉化為木糖醇(Xylitol)、阿拉伯糖醇(Arabitol)及核糖醇(Ribitol)等多種五碳糖醇之產率。於其中,該酵母菌之培養條件係添加介於2.0~4.0克/公升(g/L)範圍之醋酸於培養液中,且該培養液係可以葡萄糖、木糖或木糖水解液為碳源培養。
上述纖維原料水解液係為可選自稻稈(Rice Straw)、蔗渣(Bagasse)、芒草(Silvergrass)、狼尾草(Napiergrass)、鳳梨皮、柳枝稷(Switchgrass)、木材(Wood)及竹子(Bamboo)之生質纖維原料經前處理後所得之木糖水解液,其中,該前處理之方法係為雙軸螺旋擠壓混酸配合熱水溶洗前處理程序、稀酸催化前處理程序、酸催化蒸汽爆裂前處理程序或其他稀酸前處理程序。
當本發明於運用時,該酵母菌之培養程序係至少包含下列步驟:(A)係添加醋酸於已預先經氫氧化鈉(NaOH)調理至酸鹼值5.0之纖維原料木糖水解液中,再混合使最終含量體積佔20%之人工木糖培養液配製成馴養液,並用於培養酵母菌,俾使該馴養液之初始木糖及醋酸濃度分別控制在40g/L及2.5g/L,其中,該人工木糖培養液係為包含木糖(Xylose)、酵母抽出物(Yeast Extract)及蛋白(Peptone)之培養基;(B)在連續繼代至少5代後,以上述初始木糖維持在固定濃度值之原則下,再逐漸提高醋酸濃度,俾使該人工木糖培養液之醋酸濃度逐步提高至3.0g/L及4.0g/L;以及(C)最後以上述醋酸含量4.0g/L之經氫氧化鈉中和木糖水解液培養液中培養至少25代以上,即得一具有較佳五碳糖
醇生產能力之酵母菌株。
本發明接續將上述培養之菌株應用於纖維原料水解液。請參閱『第1圖』所示,係本發明以6%人工合成木糖溶液之五碳糖醇及酒精生成比較示意圖。如圖所示:於一較佳實施例中,係本發明利用上述方法所培養之酵母菌,於人工配製之木糖溶液中之發酵情形,其木糖初始濃度為6%(w/w),發酵溫度控制在30℃,培養箱振盪速度維持在100~150rpm,以及發酵菌株接種量與發酵液體積比為1:6(w/w)。經實驗結果顯示,其中斜線圖條為本發明培養之菌株在合理之時間內所得之木糖醇產率,明顯較控制組菌株之產率可提高2.1倍,而核糖醇則可提升1.5倍,足見酵母菌經由本發明之方法培養後,其轉化木糖為五碳糖醇生成率確實能夠有所提高。
請參閱『第2圖~第4圖』所示,係分別為本發明木糖水解液以過鹼化法調理之流程示意圖、本發明經過鹼化法去毒程序調理後之水解液組成示意圖、及本發明以過鹼化法去毒調理水解液之五碳糖醇產率示意圖。如圖所示:於另一較佳實施例中,係本發明利用上述方法所培養之酵母菌,於過鹼化法調理過之稻稈木糖水解液生成五碳糖醇之情形。其中,該過鹼化法之調理處理流程如第2圖所示,係將木糖水解液須依次進行加熱、添加過量石灰、固液分離及添加酸劑調整木糖水解液酸鹼值至弱酸性等多個步驟,同時所生成之硫酸鈣污泥尚需另行處理及處置。
另外,本實施例中之稻稈木糖水解液係以雙軸螺旋擠壓機(詳細過程請參考本申請人以中華民國專利申請第097100129號之專利案,在此不多作贅述),搭配溶洗反應槽作為前處理
設備所得之液態溶液。其反應過程中,經裁切至適當尺寸之稻稈首先以該雙軸螺旋擠壓機破壞其結構,其操作之稀酸濃度為3%(w/w),螺桿轉速為40 rpm,反應溫度在120~130℃,以及進料稻桿乾重與水溶液之比例約為50:100。當上述稻稈經擠壓機處理後,再送入溶洗反應槽,並通入適量蒸汽,使稻桿乾重與水溶液之比例降至約30:100,同時提高反應溫度至160℃,並在此高溫下蒸煮20分鐘後,隨即將反應後之稻稈及水溶液排出,再經固液分離設備將反應後之稻稈及水溶液予以分離,最後其所得之水溶液即為木糖水解液。此木糖水解液經過鹼化法調理後之水質組成如第3圖所示。
本實施例將上述木糖水解液之酸鹼值調整至5.0,發酵溫度控制在30℃,培養箱振盪速度維持在100~150rpm,以及發酵菌株接種量與發酵液體積比為1:6(w/w),其中,控制組係僅利用人工配製木糖培養液(木糖、酵母抽出物及蛋白)培養之菌株。其實驗結果如第4圖顯示,斜線圖條為本發明培養之菌株在合理之時間內所得之木糖醇產率,明顯較控制組菌株之產率可提高3.6倍,而核糖醇則可提升4.9倍,足見酵母菌經由本發明之方法培養後,於過鹼化法調理之稻稈木糖水解液之五碳糖醇生成率確實能夠大幅地提高。
請參閱『第5圖~第7圖』所示,係分別為本發明木糖水解液未經去毒性調理之流程示意圖、本發明經氫氧化鈉中和程序調理後之水解液組成示意圖、及本發明以氫氧化鈉中和調理水解液之五碳糖醇產率示意圖。如圖所示:於再一較佳實施例中,係本發明將稻稈水解液直接添加氫氧化鈉,使水解液之酸鹼值調整至5.0,如第5圖所示。此未去毒性調理之步驟雖然
較為單純,僅需添加鹼劑將木糖水解液之酸鹼值由酸性調整至弱酸性,惟,因水解液中會含有較高濃度之糠醛及硫酸根離子等發酵抑制物,故其木糖發酵技術之困難度相對提高。
本實施例之水解液組成如第6圖所示,在進行發酵實驗時,先將溫度控制在30℃,培養箱振盪速度維持在100~150rpm,以及發酵菌株接種量與發酵液體積比為1:6(w/w),其中,控制組係僅利用人工配製木糖培養液(木糖、酵母抽出物及蛋白)培養之菌株。其實驗結果如第7圖顯示,斜線圖條為本發明培養之菌株在合理之時間內所得之木糖醇產率,明顯較控制組菌株之產率可提高5.5倍,而阿拉伯糖醇可提高3.8倍,至於核糖醇則可提升5.5倍,足見酵母菌經由本發明之方法培養後,於未去毒性調理之稻稈木糖水解液之五碳糖醇生成率確實能夠大幅地提高,且較應用於過鹼化法調理之稻稈木糖水解液發酵時之提升效果更為顯著。
藉此,經本發明以上述各實施例將前述培養後之菌株應用於纖維原料水解液之結果,可知本發明其提升纖維原料水解液發酵轉化五碳糖醇效率之應用情形。其中以未經去毒性處理之水解液為原料,其提升五碳糖醇生產效率之幅度會較經過鹼化法處理之水解液之效果為佳,據此特點將可省略水解液必須先行去毒化調理之需求,進而能相對降低所需之設備投資成本。此外,經本發明之方法培養後之酵母菌除了具有生成木糖醇、阿拉伯糖醇及核糖醇等多種五碳糖醇之能力外,尚可同時將木糖轉化為酒精,後續可藉由適當之酒精純化及濃縮技術,併同提供可作為車用燃料之纖維酒精使用。
綜上所述,本發明係一種提升纖維水解液生產五碳糖醇之
酵母菌培養方法,可有效改善習用之種種缺點,係以特定條件培養酵母菌Pichia stipitis
為發酵菌株,應用於未經去毒性處理或經過鹼化法處理之纖維原料水解液中,可提升該纖維原料水解液中之木糖轉化為五碳糖醇之產率,進而使本發明之產生能更進步、更實用、更符合使用者之所須,確已符合發明專利申請之要件,爰依法提出專利申請。
惟以上所述者,僅為本發明之較佳實施例而已,當不能以此限定本發明實施之範圍;故,凡依本發明申請專利範圍及發明說明書內容所作之簡單的等效變化與修飾,皆應仍屬本發明專利涵蓋之範圍內。
第1圖,係本發明以6%人工合成木糖溶液之五碳糖醇及酒精生成比較示意圖。
第2圖,係本發明木糖水解液以過鹼化法調理之流程示意圖。
第3圖,係本發明經過鹼化法去毒程序調理後之水解液組成示意圖。
第4圖,係本發明以過鹼化法去毒調理水解液之五碳糖醇產率示意圖。
第5圖,係本發明木糖水解液未經去毒性調理之流程示意圖。
第6圖,係本發明經氫氧化鈉中和程序調理後之水解液組成示意圖。
第7圖,係本發明以氫氧化鈉中和調理水解液之五碳糖醇
產率示意圖。
Claims (5)
- 一種提升纖維水解液生產五碳糖醇之酵母菌培養方法,係以特定條件培養酵母菌Pichia stipitis 為發酵菌株,應用於未經去毒性(Detoxification)處理或經過鹼化法(Overliming)處理之纖維原料水解液中,俾利於提升該纖維原料水解液中之木糖轉化為五碳糖醇之產率達3~6倍者;於其中,該酵母菌之培養條件係添加介於2.0~4.0克/公升(g/L)範圍之醋酸於培養液中,且該五碳糖醇係包括木糖醇(Xylitol)、阿拉伯糖醇(Arabitol)及核糖醇(Ribitol)。
- 依據申請專利範圍第1項所述之提升纖維水解液生產五碳糖醇之酵母菌培養方法,其中,該培養液係可以葡萄糖、木糖或木糖水解液為碳源培養。
- 依據申請專利範圍第1項所述之提升纖維水解液生產五碳糖醇之酵母菌培養方法,其中,該纖維原料水解液係預先添加鹼劑將酸鹼值調整至弱酸性。
- 依據申請專利範圍第1項所述之提升纖維水解液生產五碳糖醇之酵母菌培養方法,其中,該纖維原料水解液係為可選自稻稈(Rice Straw)、蔗渣(Bagasse)、芒草(Silvergrass)、狼尾草(Napiergrass)、鳳梨皮、柳枝稷(Switchgrass)、木材(Wood)及竹子(Bamboo)之生質纖維原料經前處理後所得之木糖水解液。
- 依據申請專利範圍第4項所述之提升纖維水解液生產五碳糖醇之酵母菌培養方法,其中,該前處理之方法係為雙軸螺旋擠壓混酸配合熱水溶洗前處理程序、稀酸催化前處理程序及酸催化蒸汽爆裂前處理程序。
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| Devaki, et al., "Development of xylose-fermenting yeasts for ethanol production at high acetic acid concentrations", Applied biochemistry and biotechnology, Vol. 51/52, 307-318, 1995. * |
| Huang, et al., "Enhanced ethanol production by fermentation of rice straw hydrolysate without detoxification using a newly adapted strain of Pichia stipitis", Bioresource technology, Vol. 100, 3914-3920, 5 April 2009. * |
| Tom, et al., "A rare sugar xylitol. Part I: the biochemistry and biosynthesis of xylitol", Appl. Microbiol. Biotechnol., Vol. 74, 277-281, 2007. * |
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