JP6004321B2 - Methods for controlling the growth of miscellaneous bacteria in ethanol fermentation of moss - Google Patents
Methods for controlling the growth of miscellaneous bacteria in ethanol fermentation of moss Download PDFInfo
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- JP6004321B2 JP6004321B2 JP2012094692A JP2012094692A JP6004321B2 JP 6004321 B2 JP6004321 B2 JP 6004321B2 JP 2012094692 A JP2012094692 A JP 2012094692A JP 2012094692 A JP2012094692 A JP 2012094692A JP 6004321 B2 JP6004321 B2 JP 6004321B2
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- Prior art keywords
- fermentation
- simultaneous saccharification
- antibiotic
- temperature
- moss
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- 238000000034 method Methods 0.000 title claims description 17
- 241000894006 Bacteria Species 0.000 title description 16
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- 238000010438 heat treatment Methods 0.000 claims description 18
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Images
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Description
本発明は、一般家庭などから収集されてきた厨芥類を用いたエタノール製造において、共存する雑菌の増殖を抑制して雑菌によるエタノール発酵への影響を極力及ぼさないようにする方法に関する。 The present invention relates to a method for suppressing the growth of coexisting bacteria to minimize the influence of the bacteria on ethanol fermentation in ethanol production using moss collected from general households.
産業廃棄物としての食品廃棄物は再利用が進められており、主に堆肥化、メタン発酵や、近年ではエタノール製造原料として利用されている。一方、現在焼却により処理されている一般廃棄物約３８００万トンの内およそ１５％程度を占める厨芥類（環境省ＨＰ上公開の平成１９年度統計）は、水分を多く含んでいることから焼却処理時のカロリー低下の原因ともなっており、雑多な性状と他成分との混在状態のためにエタノール製造原料に適さない場合が多く、再利用率が極めて低いのが現状である。しかし、一般廃棄物を有機資源として再利用する試みは続けられており、例えば特許文献１および２には一般廃棄物を粉砕し粉砕物から夾雑物を機械選別により除去して、残った有機性成分をメタン発酵基質として利用方法が提案されている。しかし、この方法では発酵不適物の影響により安定的な反応が困難であり、滞留時間が長いことから砂や金属などの廃棄物由来の異物の残存と反応槽の損傷などの問題がある。一方でメタンの利用に関しても問題が多く、発酵で得たメタンは発電等に利用されずに燃やされていることが多い（特許文献３）。また、複合的な菌群で構成されるメタン発酵菌のバランスが変化することにより発酵に不具合が生じた場合、大量の未発酵残渣を処理しなければならず、また再立ち上げ時に多大なコストと時間が必要である。
Food waste as industrial waste is being reused, and mainly used as composting, methane fermentation, and in recent years as a raw material for producing ethanol. On the other hand, moss (approximately 15% published on the Ministry of the Environment's website in 2007), which accounts for approximately 15% of the approximately 38 million tons of general waste currently being processed by incineration, is incinerated because it contains a lot of water. It is also a cause of calorie reduction at the time, and is often unsuitable as a raw material for producing ethanol due to the mixed state of miscellaneous properties and other components, and the reuse rate is extremely low at present. However, attempts to recycle general waste as organic resources continue. For example, in
近年，ガソリン代替に活用が求められているバイオ燃料としてバイオエタノールが注目されている。エタノール発酵はメタン発酵と比較すると滞留時間が短く、液体燃料であるため取り扱いも容易である。しかし、食品廃棄物をエタノール発酵原料とする検討は行われているものの（特許文献４）、一般廃棄物中の厨芥類を対象としたエタノール製造に関する検討は今のところ報告されていない。その理由として、一般廃棄物中の厨芥類は食品廃棄物に比べて組成が安定せず、原料中のデンプン質が豊富でなく糖化技術が確立されていないことが挙げられる。 In recent years, bioethanol has attracted attention as a biofuel that is required to be used for gasoline replacement. Ethanol fermentation has a short residence time compared to methane fermentation and is easy to handle because it is a liquid fuel. However, although studies using food waste as a raw material for ethanol fermentation have been conducted (Patent Document 4), no studies have been reported on ethanol production targeting moss in general waste. The reason is that moss in general waste is not stable in composition as compared with food waste, starch is not abundant in raw materials, and saccharification technology has not been established.
このような一般廃棄物中の厨芥類の再資源化の一手段としてこれをエタノール発酵原料に適用することは産業上有益であるが、一般廃棄物由来の厨芥類には非常に多くの微生物（雑菌）が存在する。一方で、原料としてデンプン質が多い食品廃棄物は、高温でアミラーゼを作用させて流動化することで、結果的に雑菌が少ない糖液を作ることができ、発酵反応時の滞留時間も数時間から十数時間と比較的短いため、雑菌が残存したとしても、その影響を受けにくい状態でエタノールを製造することができる。 Although it is industrially beneficial to apply this to ethanol fermentation raw materials as a means of recycling moss in general waste, a great number of microorganisms ( Miscellaneous bacteria). On the other hand, food waste with a high starch content as a raw material can be made into a sugar solution with few miscellaneous bacteria by fluidizing it by the action of amylase at a high temperature, and the residence time during fermentation reaction is several hours. Therefore, even if miscellaneous bacteria remain, ethanol can be produced in a state where it is not easily affected.
一般廃棄物中の厨芥類を発酵原料とした場合にはデンプン質が少なく、一方で多くのセルロース成分が含まれ、セルラーゼを加えて同時糖化発酵反応によりエタノールを製造する方法が適していると考えられる。その場合反応期間が数日を要することもあり、単純な糖液の発酵より長くなることから雑菌の影響が危惧される。したがって雑菌が多く存在し反応期間が長くなるという特徴を持つ一般廃棄物由来の厨芥類を含む原料の同時糖化発酵反応における雑菌抑制方法が求められる。 When moss in general waste is used as a fermentation raw material, it is considered that a method of producing ethanol by simultaneous saccharification and fermentation by adding cellulase and containing a large amount of cellulose component while containing a large amount of cellulose is considered suitable. It is done. In this case, the reaction period may take several days, which is longer than the simple fermentation of sugar liquor, and there is concern about the effects of various bacteria. Therefore, there is a need for a method for suppressing germs in a simultaneous saccharification and fermentation reaction of raw materials containing moss derived from general waste, which has a feature that many germs exist and the reaction period becomes long.
本発明者らは、一般廃棄物中に含まれる厨芥類を用いた同時糖化発酵反応時に微生物の悪影響を抑制するという雑菌抑制方法について鋭意研究を重ねた結果、厨芥類を含む原料の低温熱処理を行ない、温度降下後に同時糖化発酵反応系に抗生剤を添加することで、雑菌の非存在下と同等のエタノール製造効率を得ることができる方法を発見した。 The inventors of the present invention have conducted extensive research on a method for suppressing the harmful effects of microorganisms during simultaneous saccharification and fermentation reactions using moss contained in general waste, and as a result, conducted low-temperature heat treatment of raw materials containing moss. In addition, the inventors discovered a method that can obtain an ethanol production efficiency equivalent to that in the absence of various bacteria by adding an antibiotic to the simultaneous saccharification and fermentation reaction system after the temperature drops.
本発明は、一般廃棄物中の厨芥類からなる発酵原料を６０℃から１００℃で２時間以上低温領域で熱処理し、得られた熱処理物を発酵微生物の生育可能温度域まで降温させ、得られた降温処理物を、糖化酵素としてセルラーゼを用いて２８から４２℃、２から７日間、同時糖化発酵反応に付してエタノールを製造するに当たり、同時糖化発酵反応系に抗生剤を添加することを特徴とする雑菌増殖抑制方法である。 The present invention is obtained by heat-treating fermentation raw materials composed of moss in general waste at a temperature range of 60 ° C. to 100 ° C. for 2 hours or more and lowering the temperature of the obtained heat-treated product to a temperature range where the fermentation microorganisms can grow. In order to produce ethanol by subjecting the temperature-decreasing treatment product to ethanol at 28 to 42 ° C. for 2 to 7 days using cellulase as a saccharification enzyme, an antibiotic is added to the simultaneous saccharification and fermentation reaction system. This is a characteristic method for suppressing the growth of various bacteria.
本明細書および特許請求の範囲を通して、一般廃棄物とは、廃棄物のうちで「廃棄物処理法（廃棄物の処理及び清掃に関する法律）」によって定義される産業廃棄物以外のものを指し、多くは一般家庭で通常の生活を営む上で排出されるごみ、すなわち家庭系一般廃棄物である。厨芥類とは、一般家庭等のから出るのくずやのなどのごみである厨芥が主構成をなすが、包装、容器、割り箸、捕集ネットなどが混在しているため、例えば厨芥が５０％以上を占めるごみを指す。厨芥が主構成をなすように一般廃棄物に選別処理を施したものが好ましい。選別方法は特段限定されるものでなく、例えば重力選別や風力選別といった選別手段が適用でき、手選別でもよい。 Throughout this specification and claims, general waste refers to waste other than industrial waste as defined by the “Waste Disposal Act (Act on Waste Disposal and Cleaning)”, Most of them are garbage that is discharged in the ordinary life of ordinary households, that is, household waste. Garbage mainly consists of garbage such as litter and garbage from ordinary households, etc., but packaging, containers, disposable chopsticks, collection nets, etc. are mixed, so for example 50% It refers to garbage that occupies the above. It is preferable that the general waste is subjected to a sorting process so that the cocoon constitutes the main component. The sorting method is not particularly limited. For example, sorting means such as gravity sorting and wind sorting can be applied, and manual sorting may be used.
本発明では、一般廃棄物中の厨芥類をエタノール発酵原料として用いる。 In the present invention, moss in general waste is used as a raw material for ethanol fermentation.
低温領域での熱処理とは、一般的な滅菌処理が１００℃以上の温度での蒸気滅菌で行われるのに対して、これより低温の温度領域での熱処理であって、好ましくは６０℃以上、さらに好ましくは７０℃以上である。低温熱処理中の熱分布は均一であることが望ましく、そのためには機械的な攪拌を伴うことが好ましい。低温熱処理の加熱手段として抽気蒸気を用いてもよい。 The heat treatment in the low temperature region is a heat treatment in a temperature region lower than this while the general sterilization treatment is performed by steam sterilization at a temperature of 100 ° C. or higher, preferably 60 ° C. or higher. More preferably, it is 70 degreeC or more. It is desirable that the heat distribution during the low-temperature heat treatment is uniform, and for this purpose, it is preferable to involve mechanical stirring. Extraction steam may be used as heating means for the low-temperature heat treatment.
低温熱処理時間は所定の温度に到達してから２時間以上が望ましく、さらに望ましくは４時間以上である。低温熱処理時間の上限は、特に定められないが、熱処理温度と関連し、経済性を考慮すると２４時間程度である。 The low temperature heat treatment time is desirably 2 hours or more after reaching a predetermined temperature, and more desirably 4 hours or more. The upper limit of the low temperature heat treatment time is not particularly defined, but is related to the heat treatment temperature and is about 24 hours in consideration of economy.
ついで、低温熱処理で得られた熱処理物を発酵微生物の生育可能温度域まで降温させる。発酵微生物の生育可能温度域は好ましくは４から４５℃、より好ましくは２８から４２℃であり、通常は常温である。 Next, the heat-treated product obtained by the low-temperature heat treatment is lowered to the temperature range where the fermentation microorganisms can grow. The viable temperature range of the fermenting microorganism is preferably 4 to 45 ° C, more preferably 28 to 42 ° C, and usually room temperature.
ついで、熱処理物の降温で得られた降温処理物を同時糖化発酵反応に付してエタノールを製造する。同時糖化発酵は、ノボザイムズ社製セルラーゼであるセリック、ジェネンコア社製セルラーゼであるアクセルレースなどの酵素と、サッカロマイセスセレビジエなどの酵母の存在下に、好ましくは２８から４２℃、２から７日間、常法に従って実施される。 Next, ethanol is produced by subjecting the temperature-lowered product obtained by the temperature-lowering of the heat-treated product to a simultaneous saccharification and fermentation reaction. The simultaneous saccharification and fermentation is preferably performed at 28 to 42 ° C. for 2 to 7 days in the presence of an enzyme such as Celic, a cellulase manufactured by Novozymes, and an accelerator such as Accel Race, a cellulase manufactured by Genencor, and a yeast such as Saccharomyces cerevisiae. Implemented according to law.
同時糖化発酵反応系に添加される抗生剤は、一般的に抗生物質といわれる物質である。抗生剤は、抗菌スペクトルが広いものほど好ましく、有効濃度が低いものほど好ましく、低コストで製造できるものほど好ましい。 The antibiotic added to the simultaneous saccharification and fermentation reaction system is a substance generally called an antibiotic. Antibiotics having a broad antibacterial spectrum are preferable, those having a low effective concentration are preferable, and those that can be manufactured at low cost are preferable.
このような抗生剤として、例えばアクテノマイシン、アジスロマイシン、アスポキシシリン、アムホテリシン、アルベカシン、アンピシリン、エリスロマイシン、オキサシリン、オキシテトラサイクリン、カナマイシン、カルベニシリン、クリンダマイシン、クロラムフェニコール、クロルテトラサイクリン、ゲンタマイシン、シクロセリン、ジヒドロストレプトマイシン、ストレプトマイシン、スペクチノマイシン、セファロスポリン、セファロチン、セファロルジン、セフキノム、タイロシン、テトラサイクリン、ナイスタチン、ネオマイシン、バージニアマイシン、ハイグロマイシン、バシトラシン、パロモマイシン、バンコマイシン、ピューロマイシン、プラスチシジン、ブレオマイシン、ペニシリン、ポリミキシン、マイトマイシン、ミコフェノール酸、メチシリン、リンコマイシンとこれら薬剤の誘導体および塩のうち、少なくとも１つが適宜使用される。 Examples of such antibiotics include actinomycin, azithromycin, aspoxicillin, amphotericin, arbekacin, ampicillin, erythromycin, oxacillin, oxytetracycline, kanamycin, carbenicillin, clindamycin, chloramphenicol, chlortetracycline, gentamicin, cycloserine, dihydro Streptomycin, streptomycin, spectinomycin, cephalosporin, cephalothin, cephaloldin, cefquinome, tylosin, tetracycline, nystatin, neomycin, virginiamycin, hygromycin, bacitracin, paromomycin, vancomycin, puromycin, plastidin, bleomycin, penicillin, polymyxin Mitmaishi , Mycophenolic acid, methicillin, of lincomycin and these drugs derivatives and salts, at least one is appropriately used.
抗生剤の添加量は、同時糖化発酵反応物全体の重量に対して好ましくは２ｐｐｍ以上である。この添加量の上限は特に定められないが、費用対効果から１００ｐｐｍ程度である。 The added amount of the antibiotic is preferably 2 ppm or more with respect to the weight of the entire simultaneous saccharification and fermentation reaction product. Although the upper limit of this addition amount is not specifically defined, it is about 100 ppm from the cost effectiveness.
抗生剤を同時糖化発酵反応の開始時に添加することも好ましい。 It is also preferable to add an antibiotic at the start of the simultaneous saccharification and fermentation reaction.
本発明により、一般廃棄物由来の厨芥類中に多く存在する雑菌が同時糖化発酵反応に及ぼす影響を効果的に抑制することができる。その結果、従来は焼却処分されるのみであった一般廃棄物中に存在する厨芥類を同時糖化発酵原料に適用する際にエタノール収率が向上し、安定的な反応を実現できる。さらに最終エタノール濃度を向上させ、蒸留に要するエネルギーコストを減少させる上に、設備コストの占める割合を減じることができる。結果として一般廃棄物中に多く存在する厨芥類という有機資源をバイオ燃料の原料として利用することができる。 According to the present invention, it is possible to effectively suppress the influence of miscellaneous bacteria present in moss derived from general waste on the simultaneous saccharification and fermentation reaction. As a result, the ethanol yield can be improved and stable reaction can be realized when the moss present in the general waste, which has been conventionally only incinerated, is applied to the raw material for simultaneous saccharification and fermentation. Furthermore, the final ethanol concentration can be improved, the energy cost required for distillation can be reduced, and the proportion of the equipment cost can be reduced. As a result, organic resources such as moss present in general waste can be used as raw materials for biofuels.
以下、実施例および図面に基づいて本発明を具体的に説明する。 Hereinafter, the present invention will be specifically described based on examples and drawings.
実施例
自治体で収集された一般廃棄物から手選別により厨芥を抽出し、これをマルチプロセッサー（ＣＨＵＢＵ製ＰＳ−３０００Ｓ）にて原型を留めなくなるまでミキシングし、得られた混合物約１００ｋｇを１０５℃で数時間乾燥させて乾燥重量％を求めたところ、３０．３９％であった。
[Example] Soot is extracted by hand sorting from municipal solid waste collected by the local government, and mixed with a multiprocessor (PS-3000S manufactured by CHUBU) until the prototype cannot be retained. About 100 kg of the resulting mixture is obtained at 105 ° C. When dried for several hours, the dry weight% was determined to be 30.39%.
得られた乾燥物に水を注ぎその割合を２５重量％に調整し、複数のサンプルを得た。各サンプルに５０，６０，７０，８０℃で、それぞれ２，４時間、低温熱処理を施した。ついで常温まで冷却後、ジェネンコア社製のセルラーゼ「アクセルレース」と酵母「サッカロマイセスセレビシエ」をそれぞれ加え、同時糖化発酵反応の際に、各反応系に抗生剤としてクロラムフェニコールを反応物全体の重量に対し５ｐｐｍ添加し、よく攪拌した後、同時糖化発酵反応を行った。 Water was poured into the obtained dried product and the ratio was adjusted to 25% by weight to obtain a plurality of samples. Each sample was subjected to low-temperature heat treatment at 50, 60, 70, and 80 ° C. for 2, 4 hours, respectively. Next, after cooling to room temperature, Cellulase “Accel Race” manufactured by Genencor and yeast “Saccharomyces cerevisiae” were added, and chloramphenicol was added to each reaction system as an antibiotic during the simultaneous saccharification and fermentation reaction. After adding 5 ppm to the mixture and stirring well, a simultaneous saccharification and fermentation reaction was performed.
比較例１
抗生剤の添加を行わなかった点を除いて、実施例と同じ条件で操作を行った。
Comparative Example 1
The operation was performed under the same conditions as in the Examples, except that no antibiotic was added.
比較例２
ポジティブコントロールとして低温熱処理を１２１℃、２０分の条件で行った点を除いて、実施例と同じ条件で操作を行った。
Comparative Example 2
As a positive control, the operation was performed under the same conditions as in the examples except that low-temperature heat treatment was performed at 121 ° C. for 20 minutes.
比較例３
ネガティブコントロールとして低温熱処理を行わずに抗生剤を添加しなかった点を除いて、実施例と同じ条件で操作を行った。
Comparative Example 3
As a negative control, the operation was performed under the same conditions as in the Examples, except that no low temperature heat treatment was performed and no antibiotic was added.
比較例４
ネガティブコントロールとして低温熱処理を行わなかった点を除いて、実施例と同じ条件で操作を行った。
Comparative Example 4
As a negative control, the operation was performed under the same conditions as in Examples, except that low-temperature heat treatment was not performed.
実施例および比較例の処理条件および反応経過２日、５日後のエタノール濃度を表１にまとめて示す。また、反応２日後と５日後のエタノール濃度を図１および図２に示す。これら結果によると、６０℃、４時間の低温熱処理を施した発酵原料に抗生物質を添加して行った同時透過発酵反応結果は、一般的な滅菌方法であるオートクレープ処理を行った発酵原料を用いた同時透過発酵反応結果とほぼ同等となり、また７０℃、２時間以上の低温熱処理を施した結果は、ポジティブコントロールの結果とほぼ同等となった。 Table 1 summarizes the treatment conditions of Examples and Comparative Examples and ethanol concentrations after 2 days and 5 days of reaction. The ethanol concentrations after 2 days and 5 days after the reaction are shown in FIGS. According to these results, the results of the simultaneous permeation fermentation reaction that was performed by adding antibiotics to the fermented raw material that had been subjected to low-temperature heat treatment at 60 ° C. for 4 hours were obtained from the fermented raw material that had been subjected to autoclaving, which is a general sterilization method. The result of the simultaneous permeation fermentation reaction used was almost the same, and the result of the low temperature heat treatment at 70 ° C. for 2 hours or more was almost the same as the result of the positive control.
抗生剤の存在による効果は、特に厨芥類を６０℃以下で熱処理した場合に顕著に現れた同時糖化発酵反応系への外部からの雑菌混入や、大規模にスケールアップした場合の機器の繋ぎ部分等での雑菌による汚染（コンタミネーション）を考慮すると、反応中の効果的な雑菌抑制のみならず、安定的な反応の継続に有効であることが分かる。 The effect of the presence of antibiotics, especially when moss is heat-treated at 60 ° C or less, is a remarkable part of the simultaneous saccharification and fermentation reaction system. In view of contamination (contamination) due to germs such as in the above, it can be seen that it is effective not only for effective suppression of germs during the reaction but also for the continuation of a stable reaction.
また、２日後のエタノール濃度（図１）に比べて５日後のエタノール濃度（図２）が低下しているサンプルがいくつかあった。これは雑菌がエタノールを資化したことや、生成した糖が雑菌により資化され、発酵用酵母がエタノールを資化したことが考えられるが、いずれにしても雑菌が存在したことが原因である。
５ｍＬの５％ＹＰＤ液体培地に厨芥溶液０．１ｍＬを加え、ここへ抗生剤としてクロラムフェニコールを０〜５ｐｐｍ添加し、温度を６０℃に調整した後、フォトレコーダーを用いてＯＤ＝６００の吸収により雑菌の増殖を観察した。その結果、図３に示すように、２ｐｐｍ以上の抗生剤濃度で良好な雑菌抑制効果が認められた。
In addition, there were some samples in which the ethanol concentration after 5 days (FIG. 2) was lower than the ethanol concentration after 2 days (FIG. 1). This may be because the miscellaneous bacteria assimilated ethanol, or the generated sugar was assimilated by the miscellaneous bacteria, and the yeast for fermentation assimilated ethanol, but in any case the miscellaneous bacteria existed. .
Claims (5)
The method according to any one of claims 1 to 4, wherein an antibiotic is added at the start of the simultaneous saccharification and fermentation reaction.
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