446576 經濟部智慧財產局員工消費合作社印製 A7 Β7 五、發明說明（ί ) 發明背景 利用天然或人工合成有機物包埋微生物或酵素之固定 化技術’由於具有微生物或酵素不易流失、沉降分離容 易、微生物或酵素用量節省及操作簡便等優點，最近頗為 蓬勃發展。目前較常使用之包埋劑有褐蕩膠（Alginate)、 聚丙稀酿胺（PAA)、聚乙烯醇（pva)及鹿角藻膠（κ- 。褐藻膠包埋法具有合成容易、對微生物 之傷害小等優點，但其擔體強度較差，尤其在含有磷酸鹽 或其他舞螯合劑之狀態下，擔體中之鈣離子將被取代造成 擔體破損。鹿角藻勝（K，Carrageenan)在40eC以下自行膝 化特性對製作過程頗為不便，此外價格昂貴為其最大缺 點，亦大大限制其應用性。聚丙烯醯胺（PAA)具有價格便 且之優點，擔趙強度亦較褐藻滕佳，但其合成原料對微生 物具有毒性，合成過程較複雜且不易作成球型顆粒為其主 要缺點。聚乙烯醇（PVA)具有價格便宜、本身不具毒性、 擔雜強度高等優點’頗適合應用於微生物固定化，惟目前 使用之方法’在製造過程中尚有若干缺點等待克服。目前 常用之聚乙烯醇固定化方法，依據擔體合成之方式可分成 冷凍回溫法、硼酸法及硼酸一磷酸法等。pVA冷凍回溫法 操作程序較複雜，製作過程所需時間很長，實際生產上亦 缺乏經濟效益。硼酸法以飽和硼酸溶液為成型液，其製作 程序簡單，所需原料價格亦不高，但製作過程需在硼酸溶 液t浸泡長達12-24小時，對微生物之傷害性頗大，且所 需時間太長’實際生產上不符經濟效益β蝴酸鱗酸法乃針 4 本紙張尺度適用中画國家標準（CNS)A4規格（210x297公釐） ^ .-------------—訂·τ-------線 y (請先閱讀背面之注意事項再填寫本頁) 446576 A7 ___B7 五、發明說明（V ) 對硼酸法改良之方法，將硼酸浸泡時間縮短，以碟酸鹽與 PVA進行脂化反應使其硬化，藉以降低硼酸對微生物造成 之傷害，其擔體之強度大致隨磷酸鹽濃度及浸潰時間增加 而增加。以硼酸一磷酸法固定化後之擔體活性較硼酸法 高，但仍無法免除硼酸對微生物之影響，且其製程需分成 二段式操作，不但增加製作之困難度亦降低其經濟效益。 此彌酸法及蝴酸一碟酸法在製作過程中會有擔體凝結成團 塊之現象’不但降低其生產良率在生產過程中亦造成不 便’大大降低其商業競爭力。 發明概述 本發明之目的即在於解決上述硼酸對微生物之毒性及 製作過程結成團塊之問題。本發明採用硝酸鹽為成型劑， 藉以避免硼酸對微生物之傷害，並添加少量褐藻膠防止 PVA成型時凝結成困塊並增加其孔隙度。此方法不僅可免 除蝴酸對微生物之傷害且製作過程無凝結成團塊之問題， 此外其操作容易，單槽即可完成，無需分成二段式且製程 成本低，為頗具潛力之新發明。 經濟部智慧財產局負工消費合作社印製 本發明之原理係利用少量添加之褐藻膠與鈣離子產生 離子架橋作用，使其在PVA擔髏表面快速形成保護膜，藉 以避免膠體碰撞接觸時結成團塊，再藉由硝酸鹽與pVA分 子作用，使膠體硬化’形成堅韌具高機械強度及彈性之固 定化擔體。 本發明所使用之PVA與褐藻膠混合溶液，其中含pva 之重量比為3-30% ’鹼化度70%以上，聚合度1000-3000 ; 5 本紙張尺度適用中S 0家棵準（CNS)A4规格（210 X 297公釐) 44657 6 經濟部智慧財產局員工消費合作杜印製 A7 _______B7________ 五、發明說明（）） 含褐荡膠之重量比為0-10%。添加褐藻膠之目的為避免擔 趙成型時凝結成團塊並增加擔體之孔隙率，添加比例愈高 成形愈容易，但成本增加且擔體強度減弱β PVA比例愈高 製成擔體之強度愈高，但其黏度亦隨之提高，混合授拌較 為不易。與微生物或酵素或觸媒混合後，較適合之PVA及 褐藻膠重量比分別為5-20%及0. 3-3% » 本發明所使用之成型液為硝酸根鹽離子與鈣離子混合 液’确酸根鹽離子濃度為1〇%-飽和，鈣離子濃度為〇_ 10%。硝酸根鹽離子最便宜之來源為硝酸鉀及硝酸鈉，鈣 離子可選擇氣化鈣、亞硫酸氫鈣及硝酸鈣等。本發明膠體 硬化所需時間為1 〇分鐘至4小時，隨成型液中确酸根鹽 離子濃度而異，硝酸根鹽離子濃度愈高，膠體硬體速度愈 快，所需成型時間愈短》 本發明其中之一之製作流程如第一圖所示。將微生物 或酵素或觸媒或其等混合物之懸浮液或乳狀物或粉末與 PVA或PVA及褐藻膠混合液混合均勻，將其置於成型液中 浸泡10分鐘至4小時，待其成型後取出沖洗即可得成 品。 本發明可有效解決硼酸對微生物毒性及製作過程中 PVA擔趙結成團塊之問題’並可將製程時間縮短至1〇 2〇 分鐘，且無需分成二段式操作，減少設置之成本。此外其 使用之原料價格便宜，操作程序容易，為極具價值之固定 化技術•本發明可適用於微生物或酵素或化學反應觸媒之 固定化’因此應用之範圍頗為廣泛，包括利用微生物或酵 6 本紙張尺度適用中國a家楳準（CNS)A4规格（210 χ 297公釐） — 111 ：r-------:-訂·τ--------線 ¥ (請先閲讀背面之注音J事項再填寫本頁) 446576 A7 B7 五、發明說明（4) 素之生化反應，如食品或醫藥之生產程序或環境之污染物 處理方面。此外應用在需觸媒催化之化學反應上，亦可將 觸媒加以固定化，以降低觸媒流失並節省觸媒之消耗量。 發明之具體實例 實例1 經濟部智慧財產局員工消費合作社印製 取PVA-褐藻膠混合液，其中PVA (驗化度99%以上， 聚合度2000 )重量比16%，褐藻膠重量比1.2%，與 //£73//抑/?打細菌懸浮液（342mg/L)等量混合均 勻。將此菌體混合液缓慢低入含50%硝酸鹽及1%氣化鈣溶 液中’浸潰1小時即可製成粒徑約4-5mm之固定化擔體。 將此固定化擔體取出以水沖洗後，取出9ml置於500ml三 角錐形瓶中’加入50ml人工廢水（硝酸鹽氮濃度200mg-N/L ’峨酸二氫_ 2g/L ’碳酸氫鈉2g/L)，於三角錐瓶内 填充氫氣後以血清瓶塞封閉瓶口，進行氫氣自營脫氮試 驗。試驗結果發現硝酸鹽氮可在四天内完全去除，且去除 速率隨時間增加而增快。以相同污泥利用硼酸—磷酸法製 成固定化擔體’硼酸浸泡時間20分鐘，1 · 〇Μ磷酸二氫鈉 浸潰1小時。進行相同之試驗，結果四天後硝酸鹽兔濃度 僅從200mg-N/L降至120mg-N/L，由此可見經本發明固定 化後之擔艘活性較硕酸一麟酸法高。此外由機械強度試驗 亦可發現’本發明製作之擔體強度明顯高於硼酸—磷酸 法。 實例2 取PVAC16%)褐藻膠（1. 2%)混合液，以i : 1與 7 本紙張尺度適用令國國家標準（CNS>A4規格（210 X 297公》) ~ ------ 4465 76 at B7 經濟部智慧財產局貝工消费合作社印製 五、發明說明（f) j/ca/i'足ei/f/OjoAi/s懸浮液混合均句，將此菌體混合 液緩慢低入含50%硝酸鹽及2%氣化鈣溶液中，浸潰1小時 製成粒徑約 4-bmm 舍 Alcaligenes eutrophus I 擔 體。製成之擔體以1公升之反應槽進行連續式試驗，擔體 填充率15%，水力停留時間1小時，進流水濃度為25mg-N/L，氫氣浪度控制在0. 2mg/L以上《反應槽啟動三天 後，總氮去除率即可達90%以上，7天後總氮去除率幾乎 可達100%。當進流水濃度提高至45mg-N/L，氫氣濃度控 制在0.5mg/L以上時，總氮去除率亦可達90%以上。此擔 體之機械強度高，且去除效率穩定，可維持長期穩定操 作*操作時間可達300天以上。 實例3 取PVA-褐藻膠混合液，其中PVA(鹼化度99%以上， 聚合度2000)重量比16%，褐藻膠重量比1. 6%，與 i/i'KersiAS 細菌懸浮液（577mg/L 乾重）等量混 合均勻。將此菌體混合液緩慢低入含50%硝酸鹽及1%氣化 鈣溶液中，浸潰1小時即可製成粒徑約4-5mm之固定化擔 體。將此固定化擔體取出以水沖洗後，取出750ml於 5000ml壓克力反應器内中，加入4250ml人工廢水（硝酸鹽 氮濃度200mg-N/L，雄酸氫二鉀2g/L，填酸二氩鉀 lg/L)，於壓克力反應器内，輔以曝氣系統以保持實驗全 程之溶氡均保持在2mg-N/L以上，進行好氧異營脫氮試 驗。試驗結果發現在足量的碳源供應之下，硝酸鹽氮可在 6小時内完全被去除，且去除速率隨時間增加而增快，最 8 本紙張^度適用>3困家標準（CNS>A4规格(210 X 297公* ) (請先閱讀背面之注意事項再填寫本頁) --------，訂 i·-------線 446576 Α7 ------- 五、發明說明（b ) 大反應速率常數以零階反應動力模擬所得到的值為16.5mg_ N/L/hr。 實例4 取PVA(16%)與褐藻膠（i.6%)之混合液，以1 : 1比例 與Citrobacter diversus懸浮液氣合均勻，依據上邋之 合成程序製成心>e/_sz/s之pvA擔體。製成 之擔體以5公升之反應槽進行連續式試驗，擔體填充率 15% ’水力停留時間4小時，進流水濃度為65mg—N/L，氧 氣濃度控制在2mg/L以上。反應槽啟動達穩定約需7天， 總氮去除率即可達60%以上’且去除率穩定，可維持長期 操作時間可達300天以上。 圖式簡單說明 第一圖為本發明製作流程之例示。 -------------f------ J'1訂·：--------線 . * . - (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製446576 Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (ί) Background of the invention The use of natural or artificial organic matter to embed microorganisms or enzymes is immobilized. The advantages of saving microorganisms and enzymes and simple operation have been booming recently. At present, the commonly used embedding agents are Alginate, Polyacrylamide (PAA), Polyvinyl Alcohol (pva), and Staghorn Algae (κ-). The alginate embedding method is easy to synthesize and is effective for microorganisms. The advantages of less damage, but the strength of the support is poor, especially in the state containing phosphate or other dance chelating agents, calcium ions in the support will be replaced and cause damage to the support. Carrageenan (K, Carrageenan) at 40eC The following self-kneeization characteristics are quite inconvenient for the production process. In addition, its high price is its biggest disadvantage, which also greatly limits its applicability. Polyacrylamide (PAA) has the advantages of being convenient and affordable, and its strength is better than brown alga. However, its synthetic raw materials are toxic to microorganisms, the synthesis process is more complicated and it is not easy to make spherical particles. Its main disadvantages. Polyvinyl alcohol (PVA) has the advantages of cheap price, non-toxicity itself, and high burden strength. However, there are still some shortcomings to be overcome in the manufacturing process. At present, the commonly used immobilization methods of polyvinyl alcohol can be divided into different methods based on the synthesis of the support. Freezing temperature method, boric acid method, boric acid monophosphate method, etc. pVA freezing temperature method is complicated in operation procedures, the production process takes a long time, and there is no economic benefit in actual production. The boric acid method uses a saturated boric acid solution as the molding solution. The production process is simple, and the price of the required raw materials is not high, but the production process needs to be soaked in boric acid solution t for up to 12-24 hours, which is quite harmful to microorganisms and takes too long. The actual production is not in line with economic benefits. β-Phosphate acid scale method is needle 4 paper size applicable to Chinese painting national standard (CNS) A4 specifications (210x297 mm) ^ .------------- Order · τ ---- --- Line y (Please read the precautions on the back before filling this page) 446576 A7 ___B7 V. Description of the invention (V) The method of improving the boric acid method, shortening the soaking time of boric acid, and fatting with dishate and PVA The reaction hardens to reduce the damage caused by boric acid to microorganisms, and the strength of its support increases with the increase of phosphate concentration and immersion time. The activity of the support immobilized by the boric acid monophosphate method is higher than that of the boric acid method, but Still unable to exempt the effect of boric acid on microorganisms And its production process needs to be divided into two-stage operation, which not only increases the difficulty of production but also reduces its economic benefits. This lysate method and phostic acid-disc acid method will cause the body to condense into agglomerates during the production process' Not only reduces its production yield, it also causes inconvenience in the production process, which greatly reduces its commercial competitiveness. Summary of the invention The purpose of the present invention is to solve the above-mentioned problems of the toxicity of boric acid to microorganisms and the formation of agglomerates during the manufacturing process. The present invention uses nitrates It is a molding agent to prevent boric acid from harming microorganisms, and to add a small amount of alginate to prevent PVA from coagulating into lumps and increasing its porosity during molding. This method not only avoids the damage caused by butterfly acid to microorganisms, but also does not coagulate into clumps during production In addition, it is easy to operate, can be completed in a single tank, does not need to be divided into two stages, and has a low manufacturing cost, which is a promising new invention. The principle of the present invention printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is to use a small amount of added alginate and calcium ions to generate an ion bridging effect, so that it can quickly form a protective film on the surface of the PVA crossbones, so as to avoid colloids from forming clumps when they collide. Block, and then through the action of nitrate and pVA molecules, the colloid is hardened to form a tough fixed support with high mechanical strength and elasticity. The mixed solution of PVA and alginate used in the present invention, the weight ratio of which contains pva is 3-30%, the degree of alkalinity is more than 70%, and the degree of polymerization is 1000-3000; ) A4 specification (210 X 297 mm) 44657 6 Consumption cooperation by employees of the Intellectual Property Bureau of the Ministry of Economic Affairs, printed A7 _______B7________ V. Description of the invention ()) The weight ratio of brown rubber is 0-10%. The purpose of adding alginate is to avoid the coagulation of the agglomerates during molding and increase the porosity of the support. The higher the addition ratio, the easier it is to form, but the cost increases and the strength of the support decreases. The higher the β PVA ratio, the stronger the strength of the support. The higher the viscosity, the higher the viscosity. After mixing with microorganisms or enzymes or catalysts, the suitable weight ratios of PVA and alginate are 5-20% and 0.3-3% respectively. »The molding solution used in the present invention is a mixed solution of nitrate ion and calcium ion. 'The acid ion concentration is 10% -saturated, and the calcium ion concentration is 0-10%. The cheapest sources of nitrate ions are potassium nitrate and sodium nitrate. Calcium ions can be selected from gasified calcium, bisulfite and calcium nitrate. The time required for the colloidal hardening of the present invention is 10 minutes to 4 hours, which varies with the concentration of acidic ion in the molding solution. The higher the concentration of nitrate ion, the faster the colloidal hardware speed, the shorter the molding time required. The manufacturing process of one of the inventions is shown in the first figure. Mix the suspension or milk or powder of microorganisms or enzymes or catalysts or their mixture with PVA or PVA and Fucoidan, and immerse them in the molding solution for 10 minutes to 4 hours. Take out and rinse to get the finished product. The invention can effectively solve the problems of boric acid's toxicity to microorganisms and the formation of agglomerates by PVA in the production process', and can shorten the process time to 1020 minutes, and does not need to be divided into two-stage operation, reducing the cost of installation. In addition, the raw materials used are cheap and easy to operate. It is a valuable immobilization technology. The invention can be applied to the immobilization of microorganisms or enzymes or chemical reaction catalysts. Therefore, the scope of application is quite wide, including the use of microorganisms or Fermentation 6 This paper size is applicable to China A standard (CNS) A4 (210 χ 297 mm) — 111: r -------:-order · τ -------- line ¥ ( Please read the note J on the back before filling this page) 446576 A7 B7 V. Description of the invention (4) Biochemical reaction of the element, such as the production process of food or medicine or the treatment of environmental pollutants. In addition, it can also be used in chemical reactions that require catalyst catalysis. The catalyst can also be immobilized to reduce catalyst loss and save catalyst consumption. Specific Examples of Invention Example 1 The PVA-alginate mixed solution was printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, where the weight ratio of PVA (more than 99% test, polymerization degree 2000) was 16%, and the algin glue was 1.2% by weight. Mix with equal amount of // £ 73 // 抑 /? Hit bacteria suspension (342mg / L). This bacterial cell mixture was slowly lowered into a solution containing 50% nitrate and 1% calcium gasified solution and dipped for 1 hour to prepare an immobilized support with a particle diameter of about 4-5 mm. After taking out this immobilized support and rinsing with water, take out 9ml and put it into a 500ml triangular conical flask. 'Add 50ml artificial wastewater (nitrate nitrogen concentration 200mg-N / L' Eric acid dihydrogen_ 2g / L 'Sodium bicarbonate 2g / L). After filling the triangular conical flask with hydrogen, the bottle mouth was closed with a serum stopper, and a hydrogen self-denitrification test was performed. The test results showed that nitrate nitrogen can be completely removed in four days, and the removal rate increases with time. The same sludge was prepared by the boric acid-phosphoric acid method to obtain an immobilized support 'boric acid soaking time for 20 minutes, and 1.0 M sodium dihydrogen phosphate was impregnated for 1 hour. The same test was carried out. As a result, after four days, the concentration of the nitrate rabbit decreased from 200 mg-N / L to 120 mg-N / L. It can be seen that the activity of the carrier after the immobilization of the present invention is higher than that of the oleic acid method. In addition, it can be found from the mechanical strength test that the strength of the carrier produced by the present invention is significantly higher than that of the boric acid-phosphoric acid method. Example 2 Take a mixture of PVAC16%) alginate (1.2%), i: 1 and 7 This paper size applies the national standard (CNS > A4 specification (210 X 297 public)) ~ ------ 4465 76 at B7 Printed by the Shellfish Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. 5. Description of the invention (f) The j / ca / i'zuei / f / OjoAi / s suspension is mixed uniformly, and this bacterial mixture is slowly lowered. Into a solution containing 50% nitrate and 2% calcium gas, immersed for 1 hour to make a Alcaligenes eutrophus I support with a particle size of about 4-bmm. The prepared support was subjected to continuous testing in a 1 liter reaction tank. The filling rate of the carrier is 15%, the hydraulic retention time is 1 hour, the influent water concentration is 25mg-N / L, and the hydrogen wave concentration is controlled above 0.2mg / L. "After three days of the reaction tank starting, the total nitrogen removal rate can reach 90 Above 7%, the total nitrogen removal rate can reach almost 100% after 7 days. When the influent water concentration is increased to 45mg-N / L and the hydrogen concentration is controlled above 0.5mg / L, the total nitrogen removal rate can also reach more than 90%. This support has high mechanical strength and stable removal efficiency, which can maintain long-term stable operation. * The operation time can be more than 300 days. Example 3 Take a PVA-alginate mixed solution, in which PVA (alkaline The chemical degree is above 99%, the polymerization degree is 2000), the weight ratio is 16%, the algin gum weight ratio is 1.6%, and it is mixed with i / i'KersiAS bacterial suspension (577mg / L dry weight) in equal amounts. The solution is slowly lowered into a solution containing 50% nitrate and 1% calcium gas, and immersed for 1 hour to make an immobilized support with a particle size of about 4-5 mm. After removing the immobilized support and washing it with water, Take out 750ml into 5000ml acrylic reactor, add 4250ml artificial wastewater (200mg-N / L of nitrate nitrogen concentration, 2g / L of dipotassium hydrogen acid, lg / L of potassium diargonate), add in acrylic In the reactor, supplemented with an aeration system to keep the dissolution rate of the whole experiment all above 2mg-N / L, the aerobic heterogeneous denitrification test was conducted. The test results found that under the supply of sufficient carbon source, nitrate Nitrogen can be completely removed in 6 hours, and the removal rate will increase with time. The maximum 8 papers are suitable for the> 3 standard (CNS)> A4 size (210 X 297 male *) (please read the back first) Please note this page before filling in this page) --------, order i · ------- line 446576 Α7 ------- V. Description of the invention (b) The large reaction rate constant is zero First order reaction The value obtained from the simulation was 16.5 mg_N / L / hr. Example 4 A mixed solution of PVA (16%) and alginate (i.6%) was used to uniformly vaporize the Citrobracter diversus suspension at a ratio of 1: 1. The above-mentioned synthesis procedure is made into a pvA carrier of the heart> e / _sz / s. The prepared support was subjected to a continuous test in a 5 liter reaction tank. The support filling rate was 15%, and the hydraulic retention time was 4 hours. The influent water concentration was 65 mg-N / L, and the oxygen concentration was controlled above 2 mg / L. It takes about 7 days for the reaction tank to start stable, and the total nitrogen removal rate can reach 60% or more ', and the removal rate is stable, which can maintain long-term operation time of more than 300 days. Brief Description of the Drawings The first drawing is an illustration of the production process of the present invention. ------------- f ------ J'1 order: -------- line. *.-(Please read the notes on the back before filling in this Page) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs