牛場廢水含有大量有機物、氮和磷等成分，直接排放會造成嚴重的優養化和環境汙染。常見的牛場廢水處理方式是進行厭氧消化，不但可以進行廢水處理，還能產生沼氣來發電，然而此方法有後續沼渣與沼液需處理的問題。因此，本研究乃應用藻菌共生系統進行牛場廢水之生物處理；主要是利用藻菌之協同作用，有效去除廢中之有機質及營養鹽，且產生之微藻生物質可進行加值利用。微藻生物質富含豐富的養分，可以轉化成更有價值的副產品，例如生質能源、飼料和肥料等，並可減少好氧處理過程所產生大量污泥而造成的二次汙染。本研究的目標是以最適化操作策略設計出一個有效的藻菌共生系統，並配合工程策略來處理能場廢水，以達到國內牛場廢水之排放標準。此外，本研究亦探討微藻和細菌的最佳共培養條件，且評估藻菌共生系統對牛場廢水處理的效率。
本研究首先探討微藻和細菌在牛糞廢水處理上分別具有的不同功能，發現微藻的生長在未滅菌的情形下明顯優於滅菌的牛糞廢水，而且在細菌的存在下能去除更多的有機物，且實驗結果顯示微藻的主要功能是去除廢水中的氮和磷。
接著，根據未滅菌的牛場廢水處理效率和微藻生長情形來篩選出適合處理牛場廢水的藻種，發現Chlorella sorokiniana AK-1在對牛場廢水之耐受性和汙染物的去除，皆有最佳的表現，且可直接培養在50%未滅菌的牛場廢水，因此降低培養微藻所需的培養基成本，以牛場廢水培養出的Chlorella sorokiniana AK-1其生物質中富含碳水化合物，可達到最高含量47.6±2.7%和最大產率17.8±2.2 g/L/d，其次分別為蛋白質、脂質和葉黃素，最高含量分別為19.1±2.5%、15.8±1.8%和1.7±0.31 mg/g，而最大產率分別是7.4±1.7 g/L/d、5.2±1.2 g/L/d和0.65±0.08 mg/L/d。
為了改善牛場廢水有機物之去除效率，透過曝氣2vvm的半間歇操作的方式和90%的高置換率來馴養牛場廢水當中的有效菌群，發現加入0.4 g/L的馴化混菌並同時降低曝氣量(0.2 vvm)可有效改善有機物的去除效率。並發現最適化微藻接種量是0.10 g/L且和0.4 g/L的馴化混菌共培養在50%未滅菌的牛場廢水，經過7天培養後，藻菌共生系統相較於純菌和純藻系統展現出最好的微藻生長情形和營養鹽移除效率，且最大藻體濃度可達4.32±0.31 g/L，而COD、BOD、TN、NH3N和TP的移除效率則分別為85.3±1.3%、98.0±0.2%、86.0±4.9%、99.6±0.0%和100.0±0.0%。
此外，本研究評估不同的微藻接種時間是否會影響牛場廢水處理，發現在第0天僅接種混菌，而於第3天再接種微藻Chlorella sorokiniana AK-1，可提升COD和TN的移除效率。相對於第0天同時接種混菌及微藻AK-1，其COD和TN的移除效率分別從80.5±2.4%和83.9±2.9%，提升到84.3±3.2%和90.2±2.4%。且於廢水處理3天後再加微藻時，總懸浮固體（TSS）和揮發性懸浮固體（VSS）在經過重力沉降2小時後可分別從1725±290 mg/L和1621±176 mg/L降低到256±72 mg/L和230±49 mg/L，可以得知藻菌的自聚集效果更加明顯，導致藻體沉降能力更好，有利於後續生物質之收集。藉由次世代定序(NGS)分析藻菌共生系統，發現存在牛場廢水中的主要菌群如下：Rhizobiaceae Sericytochromatia unclassified, Pirellula sp, Devosia unclassified, Acinetobacter unclassified, Acinetobacter towneri 及Owenweeksi。
最後，本研究採用工程策略來提高廢水汙染物移除效率和穩定性，實驗可以分成兩部分，(1) 固定化載體的添加，以及(2) 創新的兩階段程序。在固定化載體的部分，添加3 wt%活性碳可以提升藻菌共生系統對牛場廢水汙染物移除之效率。而創新二階段程序則是根據上述第三天接種C. sorokiniana AK-1來提升整體廢水處理效果，此程序分為兩個槽來縮短廢水處理的時間，且每個循環所花費的處理時間縮短為3天，第一槽的功能是利用細菌大量移除牛場廢水中的COD和BOD，之後再利用第二槽中的藻菌共生系統將TN和TP降至更低的濃度。實驗結果顯示，在最適化90%置換率和3 wt%活性碳添加的條件下，牛場廢水的負荷量可以從55.6%提升到77.8%，且COD、BOD、TN和TP的移除效率可達90-99%，且處理後的放流水符合國內牛場廢水之排放標準 (即COD < 450 mg/L、BOD < 80 mg/L)。COD、BOD、TN和TP的平均出流水含量在77.8%的廢水負荷下分別從3936±888 mg/L、1492±57 mg/L、186.6±25.6 mg/L、71.3±10.1 mg/L降到405±73 mg/L、68±8 mg/L、14.0±1.1 mg/L、1.0±0.7。
綜合上述，本研究不僅發展出有效且創新的藻菌共生廢水處理系統，且因為產生的微藻生物質具有高含量的碳水化合物，可製成肥料和生物碳來增加廢水處理額外的利益，而實現經濟循環的概念。此外，以此環境友善的藻菌共生工法進行高效率的牛場廢水處理，彰顯其永續環保的特性及未來商業化應用之潛力。

Dairy manure wastewater consists of large amounts of organic matter, nitrogen, and phosphorus. If directly discharged into the environment, it would cause serious eutrophication and environmental pollution. The most common treatment of dairy manure wastewater is anaerobic digestion (AD), which produces biogas that could be used to generate electricity. However, there are some disadvantages to AD, such as high capital costs, increased greenhouse gas emissions, and the resulted liquid effluent and sludge that require further treatment. In this study, a microalgae-bacterial consortium system was developed for wastewater treatment, because of the synergistic effects in the removal of the organic matters and nutrients and the beneficial biomass reutilization. In addition, the microalgal biomass consists of valuable components, which could be transformed into value-added products, such as bio-energy, feed additive and fertilizer. Furthermore, microalgae-mediated bioremediation alleviates the secondary pollution issues associated with aerobic bacterial processes such as sludge disposal. The purpose of this research was to design effective microalgal-bacterial consortia and an optimal operation strategy for the efficacious treatment of dairy manure wastewater to satisfy the Taiwan dairy manure wastewater discharge standard. Subsequent experiments were divided into three parts to investigate the optimal conditions for the cultivation of microalgae and bacteria as well as evaluate the pollution removal efficiency of dairy manure wastewater treatment in the microalgal-bacterial consortium.
The first part of this study explored the metabolism and growth of microalgae and bacteria in the dairy manure wastewater treatment. It was observed that the growth of microalgae in non-sterilized dairy manure wastewater was significantly better than that in sterilized dairy manure wastewater, and the presence of indigenous wastewater bacteria could enhance organic matter removal. In addition, experimental results indicated that the main function of microalgae in wastewater bioremediation was the assimilation of excess nitrogen and phosphorus in wastewater.
In the second part of this study, suitable microalgal strains were screened based on the pollutant removal efficiency and growth in non-sterile dairy manure wastewater. Among the strains tested, Chlorella sorokiniana AK-1 showed the best performance for the tolerance of dairy manure wastewater and pollutant removal. AK-1 could be directly cultivated in 50% non-sterilized dairy manure wastewater, thereby reducing the cultivation costs for the procurement of nutrients. Chlorella sorokiniana AK-1 biomass cultivated in dairy manure wastewater was rich in carbohydrates compared to lutein, proteins, and lipids. The carbohydrate content and productivity of AK-1was 47.6±2.7% and 17.8±2.2 g/L/d, respectively. Besides, the maximum content of protein, lipid and lutein were 19.1±2.5%, 15.8±1.8% and 1.7±0.31 mg/g, respectively, while the maximum productivity was 7.4±1.7 g/L/d, 5.2±1.2 g/L/d and 0.65±0.08 mg/L/d, respectively. To improve the removal efficiency of organic matter, the aerobic bacterial process was optimized with the acclimated mixed bacterial consortium. The optimal conditions were 2 vvm aeration in semi-batch operation with a 90% wastewater replacement ratio using non-sterile dairy manure wastewater. The results showed that the addition of 0.4 g/L mixed bacterial inoculum could improve the removal efficiency of organic matter and lower the aeration rate to 0.2 vvm. The optimal inoculation size for microalgae was 0.10 g/L, co-cultured with 0.4 g/L acclimated mixed bacteria in 50% non-sterilized dairy manure wastewater. After 7 days of cultivation, the microalgal-bacterial consortia exhibited the best performance for microalgal growth and pollutant removal efficiency compared to the microalgae-only and the bacteria only systems, in which maximum biomass concentration reached 4.32±0.31 g/L, and the removal efficiency of COD, BOD, TN, NH3N and TP were 85.3±1.3 %, 98.0±0.2%, 86.0±4.9%, 99.6±0.0%, and 100.0±0.0%, respectively. In addition, this study evaluated whether different microalgae inoculation times would affect the dairy manure wastewater treatment. Inoculation of the mixed bacterial culture on day 0, followed by Chlorella sorokiniana AK-1 inoculation on day 3 could increase the removal efficiency of COD and TN, which increased from 80.5±2.4% to 84.3±3.2% and from 83.9±2.9% to 90.2±2.4% compared to AK-1 inoculation on day 0. Furthermore, after 2-h settlement by gravity, the total suspended solid (TSS) and volatile suspended solids (VSS) could be decreased from 1725±290 mg/L to 256±72 mg/L and from 1621±176 mg/L to 230±49 mg/L, respectively. The self-aggregation of microalgae-bacterial consortia, which facilitated biomass harvest was promoted as well. Next-generation sequencing (NGS) analysis of the microalgal-bacterial consortia identified the predominant bacteria present in dairy manure wastewater as follows: Rhizobiaceae, Sericytochromatia unclassified, Pirellula sp, Devosia unclassified, Acinetobacter unclassified, Acinetobacter towneri, and Owenweeksia.
The third part of this study adopted engineering strategies to improve the nutrient removal efficiency and stability of wastewater treatment. The experiment could be divided into two parts: solid carrier addition and the novel two-stage process. The addition of 3 wt% activated carbon increased the pollution removal efficiency in the microalgal-bacterial symbiotic system. The novel two-stage process adopted the inoculation of Chlorella sorokiniana AK-1 on day 3 to improve the overall wastewater treatment. This process was separated into two tanks to shorten the operation time of wastewater treatment to 3 days per cycle. The first tank performed the aerobic bacterial process with a high removal efficiency of COD and BOD from dairy manure wastewater, and the second tank applied the microalgal-bacterial consortium for the removal of TN and TP. The results revealed the optimal medium replacement ratio of both tanks in each cycle could be 90%. Introducing activated carbon in the two-stage process allowed an increase in the wastewater loading from 55.6% to 77.8% and the final pollutant removal efficiency of 90% to 99%. Besides, after wastewater treatment, the discharged effluent satisfied the Taiwan dairy manure wastewater discharge standards (COD <450 mg/L, BOD <80 mg/L). The effluent of average COD, BOD, TN, and TP content from the 77.8% wastewater loading decreased from 3936±888 mg/L to 405±73 mg/L, 1492±57 mg/L to 68±8 mg/L, 186.6±25.6 mg/L to 14.0±1.1 mg/L and 71.3±10.1 mg/L to 1.0±0.7 mg/L, respectively. This research not only developed an effective and innovative microalgal-bacterial symbiotic wastewater treatment system but also enhanced the additional benefits of wastewater treatment to achieve the concept of circular economy because the high content of carbohydrates from microalgal biomass could be potentially converted into fertilizers and biochar. In addition, this system was more environmental-friendly, with a high potential for commercial applications in the future.

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