魚類膳食佔集約化水產養殖經營成本的50％以上，故飼料成本佔通常水產養殖總成本的一半以上。而食肉動物和雜食動物魚類的水產飼料所需原料中，魚粉佔市場大宗，故魚粉是水產養殖飼料的主要動物蛋白來源。然而，由於魚粉的供應不穩定以及自然資源的減少，魚粉的價格已急劇上漲。因此，迫切需要尋找替代動物蛋白的來源來替代或減少水產飼料中魚粉的使用。微藻包含具有必需氨基酸的完整蛋白質譜，其成分的組成類似於魚粉，且微藻具有極高的蛋白質產率，使其成為魚粉的理想替代品。本研究發現小球藻Chlorella sorokiniana SU-9可有效地在異養條件下於無光照的情況下利用有機碳源生長，且其蛋白質含量高，使得在異養條件下培養C. sorokiniana SU-9進行蛋白質生產具有高度潛力。此外，本研究也比較在各種條件下胺基酸含量的差異，並通過胺基酸組成分析探討異養微藻的營養價值。
本研究第一部分是建立微藻蛋白質異營生產的最適化培養條件。使用不同類型的碳源，氮源，碳濃度，氮濃度，pH值和鐵離子濃度來培養C. sorokiniana SU-9。所獲得的最佳參數如下：碳源濃度為葡萄糖10 g/L；氮源濃度為硝酸鈉1.5 g/L；不控制pH值；鐵離子濃度為46 μM。在上述最佳條件下，C. sorokiniana SU-9的生物量濃度，蛋白質含量和蛋白質產率分別達到4.14 g/L，403 mg/g和382 mg/L/d。
第二部分是降低培養成本，以糖蜜代替葡萄糖。由於糖蜜可以提供所需的養分（例如蔗糖，葡萄糖，果糖和氮，維生素和無機鹽）來支持微生物的生長，因此它是微藻生長的合適的低成本碳源和養分來源。因此，在這此研究中，廢糖蜜被用於異養微藻的培養。結果顯示，使用廢糖蜜作為有機碳源，C. sorokiniana SU-9可以在異養條件下高效率生長。此外，由於糖蜜含有其他營養成分，因此可以減少培養基營養成分的添加。在50% BG-11和1.5 g/L硝酸鈉中添加10 g/L水解糖蜜（相當於7.5 g/L還原糖）可獲得最佳條件。 C. sorokiniana SU-9的生物量濃度，蛋白質含量和蛋白質產率分別達到2.11 g/L(生物質濃度), 488.3 mg/g和201.3 mg/L/d。
最後，在最佳條件下，採用饋料批次、半批次的工程策略，對C. sorokiniana SU-9進行栽培，以提高蛋白質的產量。在饋料批次系統中，獲得了更高的生物量濃度（10.12 g/L）和較差的蛋白質產率（197.18 mg/L/d）。而在半批次系統中，當置換率為75%時，有優異的生物量生產速率（1300 mg/L/d），蛋白質生產速率(450 mg/L/d）和更高的總蛋白質產量(5118.1 mg)。最後，使用半批策略的異養培養規模擴大到5 L攪拌發酵罐，其中生物質和蛋白質的生產速率分別為2200 mg/L/d和550 mg/L/d。最後，使用替代糖蜜可以有效地將每單位蛋白質的碳源成本降低約88.6%。另外，發現微藻與魚粉和大豆相比，蛋白質中包含更高的精氨酸，賴氨酸和半胱氨酸含量，這顯示出C. sorokiniana SU-9作為飼料的優勢。

Fish meal is the primary animal protein source for aquaculture feed. However, the price of fish meal has dramatically increased due to its unstable supply and the decline in its nature resources. Thus, finding alternative sources for animal proteins to replace or reduce the use of fish meal in aqua-feeds is of urgent demand. Microalgae are rich in proteins with a complete profile of essential amino acids. The composition of microalgal protein is similar to that of fish meal, making it an ideal replacement for fish meal. Microalgae appear to be a promising source of protein due to their high protein productivity. In particular, the microalga Chlorella sorokiniana SU-9 screened in this study can effectively use organic carbon sources under heterotrophic conditions without illumination, and has a high protein content. Protein production under heterotrophic conditions is more practicable for efficient mass production of proteins in industrial processes. In this study, the production of heterotrophic proteins of C. sorokiniana SU-9 strain was examined. In addition, the differences of amino acid profiles under various conditions were compared, and the nutritional value of heterotrophic microalgae was discussed through the analysis of amino acid profiles.
The first part of this study was to establish the cultivation conditions for protein production. Different types of carbon source, nitrogen source, carbon concentration, nitrogen concentration, pH value and iron ion concentration were evaluated for the cultivation of C. sorokiniana SU-9. The defined parameters for optimal protein production under heterotrophic cultivation of SU-9 are as follows: carbon source - glucose concentration of 10 g/L, nitrogen source - sodium nitrate concentration of 1.5 g/L, pH value not controlled and iron concentration of 46 μM. Under the above optimal conditions, the biomass concentration, protein content and protein productivity of C. sorokiniana SU-9 reached 4.14±0.20 g/L, 403±33 mg/g and 382±36 mg/ L/d, respectively.
In order to reduce the microalgae cultivation cost, glucose was replaced with molasses as a carbon source in the heterotrophic cultivation. As molasses wastewater can provide the required nutrients (such as sucrose, glucose, fructose, and nitrogen, vitamins and inorganic salts) to support the growth of microalgae, it is a suitable low-cost source of carbon and nutrients. Therefore, in this study, waste molasses was used for the cultivation of heterotrophic microalgae. The results show that using waste molasses as an organic carbon source, C. sorokiniana SU-9 can grow efficiently under heterotrophic conditions. Since molasses contains additional nutrients, the nutrients present in the medium for cultivation can be reduced. The results indicated that 50% BG-11, 1.5 g/L sodium nitrate, 10 g/L hydrolyzed molasses (equivalent to 7.5 g/L reducing sugar) can achieve the best protein production. The biomass concentration, protein content and protein productivity of C. sorokiniana SU-9 reached 2.11±0.31 g/L, 488±19 mg/g and 201±25 mg/L/d, respectively.
After arriving at the optimal conditions for protein production using SU-9, engineering strategies such as fed-batch and semi-batch operation were applied for the improvement of protein production. In the fed-batch system, higher biomass concentration (10.12 g/L) and poor protein productivity (211 mg/L/d) were obtained. In the semi-batch system, with a replacement rate of 75%, excellent biomass productivity (1300 mg/L/d), protein productivity (450 mg/L/d) and higher total protein accumulation of 5118.1 mg were achieved. Finally, heterotrophic culture using a semi-batch strategy was scaled up to a 5 L stirred tank fermentor, in which biomass productivity and protein productivity of 2200 mg/L/d and 550 mg/L/d, respectively were achieved. Cost analysis revealed that the use of molasses as an alternative carbon and nutrient source can effectively reduce the carbon source cost per kilogram of protein by 88.6%. In addition, it was found that compared with fishmeal and soybeans, microalgal proteins contained higher arginine, lysine and cysteine content, which exhibis the advantages of C. sorokiniana SU-9 as a feed.

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