实验海洋生物学重点实验室知识库

我国海洋红藻资源丰富，人工栽培红藻产量位居世界第一，然而红藻天然产物的高效利用仍是一个亟待解决的问题。坛紫菜及细基江蓠是我国重要的栽培经济物种，不仅富含藻胆蛋白，还含有琼胶等高值产物。本论文以坛紫菜及细基江蓠为研究对象，综合提取两种海藻的藻红蛋白和琼胶，以达到充分利用生物质、节约资源及减少废液排放量的目的。此外，利用大型绿藻浒苔对综合提取过程中产生的废液进行生物处理，优化了工艺条件，明确了原废液最佳稀释倍数、处理天数及最佳料液比。结果表明这种环境友好型的生物处理方法可有效地去除废液中的营养元素，大大节约了废液处理成本，对环境保护有着重要的指导意义，具体研究结果如下：

1. 坛紫菜及细基江蓠藻红蛋白的提取、分离和纯化

首先从1000克细基江蓠以及100克坛紫菜(鲜重)中提取、分离以及纯化藻红蛋白。利用先进的膨化床吸附技术，每克坛紫菜中可分离0.74 mg的食品级藻红蛋白（纯度＜4）。通过阴离子交换的方法，进一步纯化得到纯度大于4的分析级藻红蛋白0.24 mg，得率为0.24 mg/g。基于同样的方法从每克细基江蓠(鲜重)中可分离得到81.10 μg的食品级藻红蛋白。经纯化从每克细基江蓠（鲜重）中得到纯度大于4的分析级藻红蛋白30.30 μg。采用全波长扫描光谱、SDS-PAGE及Native-PAGE的方法对两种藻红蛋白进行纯度鉴定，结果表明利用该方法可以规模化制备出两种高纯度的藻红蛋白，通过光谱鉴定均为R-型藻红蛋白。

2. 两种海藻藻渣的琼胶提取

利用提取藻红蛋白后的坛紫菜及细基江蓠的藻渣提取琼胶，琼胶的得率分别为5.82 ± 0.17%和21.30 ± 2.00%，与直接从同等质量的两种海藻中提取琼胶的得率比较，并无显著性差异(p＞0.05)，也就是说提取藻红蛋白并不会影响后续琼胶的得率。经过一系列理化性质分析，结果显示综合提取的琼胶凝胶强度分别为796.00 ± 28.10g/cm²和789.60 ± 34.80g/cm²，凝固点分别为38.10 ± 0.22 ℃和36.20 ± 0.30 ℃，融化点为89.50 ± 0.55 ℃和91.00 ℃，3.6-内醚半乳糖含量为34.00 ± 3.16%和34.10 ± 2.00%，硫酸根含量为3.01 ± 0.24%和4.90 ± 0.30%，与直接从两种海藻中提取的琼胶相比无显著性差异(p＞0.05)，均符合工业化产品要求。另外，与采用同等质量的原藻烘干直接制备琼胶相比，综合提取法较直接提取法碱用量和废液量少，每100 g坛紫菜和1000 g细基江蓠的碱用量分别减少0.80 L和1.40 L，水用量分别减少了75.40 L和17.40 L，废液总量分别减少78.31 L和21.50 L。由此可见，利用综合提取法生产琼胶既可减少原料成本，节约水资源，又可降低后续废液处理压力。为检测所获琼胶的实际应用价值，对综合提取的琼胶进行DNA凝胶电泳分析，结果显示50 bp及2000 bp的DNA marker条带均可以清晰分离。其次，细菌培养试验结果表明提取琼胶对细菌的生长无抑制且长势良好，菌落数分别为72.30 ± 5.21以及78.20 ± 8.80个，与市售琼胶效果无显著性差异(p＞0.05)。

 3. 综合提取废液的生物处理

收集综合提取藻红蛋白及琼胶过程中产生的废液，利用浒苔对废液进行生物处理，并优化了处理条件。将废液稀释2¹、2²、2³、2⁴、2⁵、2⁶以及2⁷倍，利用浒苔处理所有废液2天，以浒苔的光合作用参数为浒苔生长情况参考依据，确定浒苔存活的最佳稀释倍数。以废液指标、浒苔的光合作用相关参数以及处理废液后的浒苔的生长率为依据，将浒苔处理废液的培养时间设置为1-7天，料液比分别设置为3.7g:1L、7.4 g:1 L、14.8 g:1 L及29.6 g:1 L，确定浒苔处理废液的最佳时间及料液比。结果表明，浒苔处理坛紫菜废液的最佳稀释倍数为2倍，在料液比7.4 g:1 L条件下处理2天效果最佳。BOD₅、COD、总氮、无机氮、氨氮、硝酸盐、亚硝酸盐、总磷以及无机磷的清除率分别为53.29 ± 0.85 %、49.16 ± 0.31%、42.50 ± 6.70%、66.53 ± 0.78%、48.59 ± 1.26%、73.47 ± 1.57%、73.59 ± 1.33%、69.30 ± 4.09% 及77.69 ± 4.54%，浒苔生长率为8.33±0.31%。同理，浒苔处理细基江蓠废液最佳稀释倍数为4倍，最佳处理天数为2天，最佳料液比与7.4:1，与处理坛紫菜综合提取的废液结果较为一致。BOD₅、COD、总氮、无机氮、氨氮、硝酸盐、亚硝酸盐、总磷以及无机磷的清除率分别为50.27 ± 1.52 %、34.02 ± 3.36%、41.34 ± 4.16%、67.30 ± 2.43%、48.53 ± 3.20、74.11 ± 7.11、76.50 ± 2.35、63.71 ± 2.54及76.30 ± 0.30，浒苔生长率为6.63±0.64%。因此，浒苔能有效清除废液中的氮磷元素，可大大减少废液外排对环境造成的污染，为相关生产企业的废液处理工作提供了理论依据。

综上所述，本论文通过综合提取两种经济海藻的藻红蛋白和琼胶，建立了综合提取方法技术体系，为生物质的高值化利用奠定了理论基础。同时，利用浒苔对综合提取产生的废液进行生物处理，可有效解决琼胶生产产业面临的废液处理问题，为经济红藻产业的高值化利用及可持续发展提供了方法和思路。

The resources of red seaweeds are abundant and distribute widely. China is the country with the largest amounts of red seaweeds in the whole world. Many researches focus on the resources of red algae, aquaculture and its uses increasingly. But making full use of red algae about the extraction of natural products is still a key problem. Pyropia haitanensis and Gracilaria tenuistipitata are the large-scale economic seaweeds that widely cultivated along the Chinese coastal areas. The two kinds of algae are not only rich in phycobiliproteins, but also other nature products with high value such as agar. In this paper, in order to make full use of Pyropia haitanensis and Gracilaria tenuistipitata, save resources and reduce the wastewater, R-phycoerythrin and agar with high additional value are extracted from the two kinds of algae successfully. In addition, the wastewater from the whole extraction are treated by green tide of Ulva prolifera. And the conditions of the treatment process were optimized to determine the optimal dilution multiple, the best treatment days and the best material-liquid ratio. This environmentally-friendly biological treatment method can remove nutrients effectively. It can save the cost of wastewater treatment and protect the environment.The main conclusions are as following:

1. Firstly, the R-phycoerythrin was extracted, isolated and purified from the two algae successfully. 0.74g/mg and 81.1μg/g of food-grade R-phycoerythrin were gained from 100.00 g of Py. haitanensis and 1000.00 g of G. tenuistipitata respectively by expended bed chromatography. The quanlity and recovery of R-phycoerythrin was 0.24g/mg and 12.12% with the purity above 4 from Py. haitanensis by ion exchange column chromatography. While the quanlity and recovery was 45.30 μg/g and 20.70% with the purity above 4 from G. tenuistipitata. And the R-phycoerythrin was then identified by SDS-PAGE, Native-PAGE and absorption spectrum. Thus, the high-value product of R-phycoerythrin was gained for large scale from these algae.

2. Secondly, the agars were extracted from the residues after the purification of R-phycoerythrin from the two species of algae. The agar yields of G.tenuistipitata and Py. haitanensis were 21.32 ± 1.95 % and 5.82 ± 0.17 % respectively. The agar from Py. haitanensis was with a gel strength of 796.00 ± 28.10 g/cm², a gelling temperature of 38.10 ± 0.22°C, a melting temperature of 89.50 ± 0.55°C, 3, 6-anhydrogalactose content of 34.00% ± 3.16%, and sulfate content of 3.01% ± 0.24%. And those data from G. tenuistipitata was 789.60 ± 34.77%, 36.20 ± 0.27%, 91.00%, 4.93 ± 0.26 and 34.07 ± 1.96% respectively. There were no significant differences between these parameters and those from the direct extraction of the two species of the algae. And the couple of physico-chemical properties were shown that the agars from these two kinds of seaweeds had the industrial applications for large-scale. In addition, the method of extraction could reduce lots of water, amount of sodium hydroxide, decrease the production of wastewater. The water consumption of the comprehensive extraction from 100.00g of Py. haitanensis or 1000.00g of G.tenuistipitata were 75.40 L or 17.40L less than the control. The consumption of alkali were 0.80L or 0.40 L less than the control. And the whole waste liquid output were 78.31L and 21.50L L less than the control. So, the method was benefit to the agar industries. Meanwhile, the four kinds of agar from two different algae could also be used as a material for DNA electrophoresis or a bacterial culture medium, and the commercial agarose or agar were as control. Based on the results of DNA electrophoresis, the bands were separated clearly  through the four kinds of agars which we extracted from G. tenuistipitata and P. haitanensis. The two kinds of agars were also used as a material for culturing the bacterial, the amounts of bacterial colonies were 78.20 ± 8.76 % and 79.40 ± 11.33% from G. tenuistipitata. And the amount of bacterial colonies were 72.30 ± 5.21 % and 70.50 ± 6.84 % from aging Py. haitanensis. The result indicated that the four types of agars from Py. haitanensis and G. tenuistipitata did not appear to show a general inhibitory effect on bacteria growth. Thus, the agar we extracted could also be applied as the common study without purification.

3. The condition of culturing the U. prolifera for treatment of wastwater from the whole process of extraction were optimized.The wastewater from the whole extraction process of Py. haitanensis or G.tenuistipitata need be diluted two times or four times. The different days were set as 1-7 days and the different material-to-liquid ratio were set as 3.7g:1L、7.4 g:1 L、14.8 g:1 L and 29.6 g:1 L respectively. The optimum time of culturing the U. prolifera for treatment of wastewater from the extraction of agar either from Py. haitanensis or G.tenuistipitata were all 48h under material-to-liquid ratio of 7.4 g:1 L. The growth rate of U. prolifera were 8.33 ± 0.31% or 6.63 ± 0.64% respectively. The results showed that the removal rates of BOD₅, COD, total nitrogen, inorganic nitrogen, ammonia nitrogen, nitrate, nitrite, total phosphorus and inorganic phosphorus were 53.29 ± 0.85%, 49.16 ± 0.31%, 42.50 ± 6.70%, 66.53 ± 0.78%, 48.59 ± 1.26%, 73.47 ± 1.57%, 73.59 ± 1.33%, 69.30 ± 4.09% and 77.69 ± 4.54% after the treatment of the wastewater from the whole extraction of Py. haitanensi for 48 hours under the material-to-liquid ratio of 7.4 g:1L. And those indicators from G. tenuistipitata were 50.27 ± 1.52 %、34.02 ± 3.36%、41.34 ± 4.16%、67.30 ± 2.43%、48.53 ± 3.20%、74.11 ± 7.11%、76.50 ± 2.35%、63.71 ± 2.54% and 76.30 ± 0.30% respectively. Thus, U. prolifera could remove N and P from the wastewater effectively. It greatly reduced the pressure on the environment caused by the effluent discharge, and provides new ideas for the wastewater treatment of related industries.

In summary, this paper establishes a comprehensive extraction technology through the inferior Py. haitanensis and G. tenuistipitata. And it lays a foundation for the full utilization of biomass. At the same time, this paper was also focused on the treatment of the wastewater from comprehensive extraction. The cheaper and simpler operation of biological treatment ws carried out by U. prolifera. It not only achieved the purpose of utilizing U. prolifera during the green tide period, but also solves the problems of wastewater treatment in the production of agar. It provided a metod of sustainable development for industries from the economic red alga that conformed to the transformation of new and old kinetic energy in China.