工业生物燃气研究组知识库

本论文的研究集中于两个方面：（1）绿藻氢化酶氧耐受性筛选研究；（2）光照绿藻产氢研究。具体研究内容如下：

对5种不同的绿藻氢化酶的氧耐受性进行了测定，结果表明：不同绿藻氢化酶氧耐受性有较大差别，其中蛋白核小球藻（Chlorella pyrenoidosa）氢化酶体内、体外氧耐受性分别比莱茵衣藻（Chlamydomonas reinhardtii CC124）高139%和61%，且在光照条件下，由于蛋白核小球藻氢化酶的氧耐受性较强，因此导致其产氢持续时间较莱茵衣藻的产氢持续时间长。

对比研究了不同条件下莱茵衣藻和蛋白核小球藻光照产氢过程。在常规光照条件下，光合放氧会严重抑制产氢。在光暗循环条件下，光照下失活的氢化酶可在黑暗条件下恢复活性，但在光照条件下迅速失活，导致莱茵衣藻和蛋白核小球藻光照产氢持续时间较短。在无硫培养条件下，莱茵衣藻产氢可持续96 h以上，而蛋白核小球藻则不能产氢。

对蛋白核小球藻在不同乙酸浓度和不同光照强度下的呼吸速率及光合放氧速率进行了测定，表明蛋白核小球藻细胞的呼吸耗氧速率随着乙酸浓度的增大而增大，而光合放氧速率则随着光照强度的降低而降低，通过优化乙酸浓度和光照强度可使蛋白核小球藻在有硫条件下达到厌氧，并实现持续产氢。

对蛋白核小球藻进行了有硫和无硫培养，并对其低光强重复产氢进行了研究，结果表明重复产氢的方法可提高蛋白核小球藻的总累积产氢量。在有硫产氢条件下，蛋白核小球藻停止产氢后，可在加入乙酸钠培养后重复产氢；在无硫产氢条件下，蛋白核小球藻可在培养1 d后产氢，且经重复培养一次后，第二次产氢阶段的蛋白核小球藻的单次产氢量仍比第一次高，说明蛋白核小球藻在有硫和无硫产氢条件下存在两种不同的代谢途径。

对蛋白核小球藻在污水（生活污水和沼液）中的生长及产氢特性进行了研究，表明蛋白核小球藻在污水和沼液中均有良好的适应性。蛋白核小球藻对25%沼液的COD、氨态氮和总磷的去除率分别达到67%、97%和98%。研究发现，蛋白核小球藻可以在沼液中添加乙酸钠直接产氢，最高产氢量达到70 mL·L^-1，而在未添加乙酸钠的条件下，产氢量仅为16 mL·L^-1。另外在添加乙酸钠培养1 d的条件下蛋白核小球藻的产氢量可大幅提高，最高产氢量达150 mL·L^-1。对不同培养条件下的蛋白核小球藻的产氢进行了比较研究，发现蛋白核小球藻在沼液中以CO₂辅助培养条件下的产氢量显著高于同条件下的生活污水培养的蛋白核小球藻，产氢量达到130.9 mL·g^-1藻干重，说明利用沼液培养蛋白核小球藻并产氢具有可行性。

This thesis focused on two aspects: (1) O₂ tolerant green algae hydrogenase screening; (2) H₂ production of green algae under illumination. The main contents of thesis are as follows:

The hydrogenase O₂ tolerance of five different green algae was investigated; it was found that the hydrogenase O₂ tolerance was different for different green algae. C. pyrenoidosa hydrogenase O₂ tolerance (vivo and vitro) was 139% and 61% higher than that of C. reinhardtii, respectively; furthermore, this feature can benefit the photo H₂ production. The duration of H₂ production of C. pyrenoidosa was longer than C. reinhardtii.

H₂ production of C. reinhardtii and C. pyrenoidosa was comparatively investigated under different condition; it was found that H₂ production was inhibited by photosynthetic O₂ evolution under normal light condition. For H₂ production under light/dark cyclic condition, the hydrogenase activity could be recovered during dark condition, but it was inactivated by photosynthetic O₂ evolution under illumination, which leading to a short period H₂ evolution. Under sulfur-deprived condition, the duration of H₂ evolution of C. reinhardtii could sustain 96 h; however, C. pyrenoidosa could not release H₂.

The respiratory and photosynthetic O₂ evolution rate of C. pyrenoidosa under different acetate concentrations and light intensities was investigated. It was found that the respiratory rates increased with the increasing of acetic acid concentrations, and photosynthetic O₂ evolution rates decreased with the decreasing of light intensities. The anaerobic environment could be obtained through optimizing the acetate acid concentrations and light intensities in sulfur replete medium. Thus, C. pyrenoidosa could sustain releasing H₂ gas under this condition.

The repeated H₂ production of C. pyrenoidosa was investigated, it was found that C. pyrenoidosa could re-produce H₂ under sulfur replete and sulfur-deprived condition; the volume and duration of H₂ production of C. pyrenoidosa can be increased by the repeated H₂ production method. Under sulfur deprived condition, C. pyrenoidosa can re-produce H₂ after one day cultivation without sodium acetate addition, and the H₂ yield increased with the increasing of repetition times, which indicating that there are two different metabolic pathways for C. pyrenoidosa under sulfur-deprived and sulfur replete conditions.

The cultivation and direct H₂ production of C. pyrenoidosa in wastewater (municipal wastewater and biogas slurry) was investigated. It was found that C. pyrenoidosa had good adaptability in wastewater。The removal efficiency of COD, ammonia nitrogen and total phosphorus were 67%, 97% and 98% respectively for biogas slurry. It was found that C. pyrenoidosa could release H₂ without replace medium in biogas slurry. The H₂ production could be increased from 16 mL·L^-1to 70 mL·L^-1 after acetate added into medium，In addition, after 24 hours of cultivation with sodium acetate , the H₂ production of C. pyrenoidosa sharply raised to 150 mL·L^-1.

The H₂ production of C. pyrenoidosa in municipal wastewater and biogas slurry was comparatively investigated; it was found that the maximum H₂ production of C. pyrenoidosa, which was cultivated in 25% biogas slurry with supply of 2% CO₂, was much higher than C. pyrenoidosa, which was cultivated in municipal wastewater with supply of 2% CO₂. The maximum H₂ production of C. pyrenoidosa was 130.9 mL·g^-1 dry weight in biogas slurry, indicating that the method of cultivation and H₂ production for C. pyrenoidosa in biogas slurry was feasible.