Biochemical Engineering Journal
生化工程学报
Volume 151, 15 November 2019, 107319
第 151 卷，2019 年 11 月 15 日，编号 107319
第 151 卷，2019 年 11 月 15 日，编号 107319
Regular article 常规文章Non-sterile heterotrophic cultivation of native wastewater yeast and microalgae for integrated municipal wastewater treatment and bioethanol production
非无菌异养培养本地污水酵母和微藻，用于综合市政污水处理和生物乙醇生产
Full text access 全文访问
Highlights 亮点
- •Microalgae growth is unsustainable under non-sterile heterotrophic conditions.
在非无菌异养条件下，微藻的生长不可持续。 - •Yeast achieved high levels of nutrient removal in non-sterile municipal wastewater.
酵母在非无菌市政废水中实现了高水平的营养物去除。 - •Crabtree positive yeast produced ethanol whilst treating municipal wastewater.
**蟹树效应阳性酵母在处理市政废水时能够产生乙醇。** - •Wild yeast achieved 92.6% orthophosphate, 100% nitrate and 100% ammonia removal.
**野生酵母能够去除 92.6%的正磷酸盐、100%的硝酸盐和 100%的氨氮。**
Abstract 摘要
Currently over 80% of wastewater generated globally, is discharged into surface waters without adequate treatment. Major environmental and public health risks associated with such releases are particularly prevalent in developing countries where the infrastructure and financing for effective treatment is lacking. Novel low cost integrated wastewater treatment and biorefinery processes could provide a sustainable solution. This study investigated, for the first time, the feasibility of simultaneous wastewater treatment and bioethanol production in non-sterile, heterotrophic bioreactors using either microalgae, wild yeast, or a co-culture of microalgae and wild yeast. Scenedesmus sp. are known to achieve high biomass concentrations under sterile heterotrophic conditions. However, under the non-sterile conditions proposed, relatively low nutrient removal rates (60% nitrate, 53% total ammonia nitrogen (TAN) and 46% orthophosphate) and biomass yields (0.98 ± 0.10 g/L) were achieved. Wastewater grown microalgae and yeast co-cultures achieved high nutrient removal rates (96% nitrate, 100% TAN and 93% orthophosphate). Wastewater grown yeast cultures produced consistently promising results, achieving high biomass concentrations of 3.7 ± 0.1 and 4.2 ± 0.1 g/L along with 100% nitrate, 100% TAN and 92.6% orthophosphate removal. Yeast provided the additional advantage of aerobic fermentation, possibly allowing integrated wastewater treatment and bioethanol production.
目前，全球超过 80%的废水未经充分处理就直接排放到地表水中。这种排放带来的主要环境和公共健康风险在发展中国家尤为突出，因为这些国家缺乏有效处理废水的基础设施和资金支持。开发新型低成本的综合废水处理和生物炼制工艺可能提供一种可持续的解决方案。本研究首次探讨了在非无菌环境下，利用微藻、野生酵母或微藻与野生酵母的共培养体系，在异养生物反应器中同时进行废水处理和生物乙醇生产的可行性。 研究表明，**Scenedesmus 属微藻**在无菌异养条件下可达到较高的生物量浓度。然而，在本文提出的非无菌条件下，其营养物去除率及生物量产量相对较低（硝酸盐去除率为 60%，总氨氮（TAN）去除率为 53%，正磷酸盐去除率为 46%，生物量产量为 0.98 ± 0.10 g/L）。相比之下，废水培养的微藻与酵母共培养体系表现出较高的营养物去除率（硝酸盐去除率为 96%，TAN 去除率为 100%，正磷酸盐去除率为 93%）。 废水培养的单一酵母体系也表现出稳定且令人满意的结果，其生物量浓度分别达到 3.7 ± 0.1 g/L 和 4.2 ± 0.1 g/L，同时实现了 100%的硝酸盐、100%的 TAN 和 92.6%的正磷酸盐去除率。酵母还具有好氧发酵的附加优势，这可能使废水处理与生物乙醇生产的集成成为可能。
目前，全球超过 80%的废水未经充分处理就直接排放到地表水中。这种排放带来的主要环境和公共健康风险在发展中国家尤为突出，因为这些国家缺乏有效处理废水的基础设施和资金支持。开发新型低成本的综合废水处理和生物炼制工艺可能提供一种可持续的解决方案。本研究首次探讨了在非无菌环境下，利用微藻、野生酵母或微藻与野生酵母的共培养体系，在异养生物反应器中同时进行废水处理和生物乙醇生产的可行性。 研究表明，**Scenedesmus 属微藻**在无菌异养条件下可达到较高的生物量浓度。然而，在本文提出的非无菌条件下，其营养物去除率及生物量产量相对较低（硝酸盐去除率为 60%，总氨氮（TAN）去除率为 53%，正磷酸盐去除率为 46%，生物量产量为 0.98 ± 0.10 g/L）。相比之下，废水培养的微藻与酵母共培养体系表现出较高的营养物去除率（硝酸盐去除率为 96%，TAN 去除率为 100%，正磷酸盐去除率为 93%）。 废水培养的单一酵母体系也表现出稳定且令人满意的结果，其生物量浓度分别达到 3.7 ± 0.1 g/L 和 4.2 ± 0.1 g/L，同时实现了 100%的硝酸盐、100%的 TAN 和 92.6%的正磷酸盐去除率。酵母还具有好氧发酵的附加优势，这可能使废水处理与生物乙醇生产的集成成为可能。
Graphical abstract 图示摘要
Keywords 关键词
Wild yeast
Sewage treatment
Non-sterile microalgae growth
Nitrate removal
Phosphorous removal
Crabtree positive yeast
野生酵母 污水处理 非无菌微藻生长 硝酸盐去除 磷去除 克拉布树效应阳性酵母
1. Introduction 1. 引言
Inadequate sewage treatment and its negative effects on health, economy and the environment is a major challenge for developing countries. Although representative data on the global generation, collection and treatment of wastewater is not well published; The United Nations, 2017 [1] estimate that over 80% of all collected wastewater is discharged without adequate treatment. The release of untreated water is particularly prevalent in developing countries where infrastructure, technical resources and financing for effective treatment is lacking. Such is the case in Mexico, where only 47.5% and 28.8% of municipal and industrial wastewater were treated in 2012 [2]. In an attempt to resolve this, a nation-wide water program was established to improve access to high quality drinking water. The program aims to prioritise individuals within the most vulnerable populations through the modernisation of wastewater treatment plants [2]. However, the high cost of operating wastewater treatment plants has hindered progress to date. The adoption of novel technologies that generate revenue from the waste could provide a sustainable solution.
发展中国家面临污水处理不足及其对健康、经济和环境造成负面影响的重大挑战。尽管关于全球污水产生、收集和处理的代表性数据尚未广泛发布，但联合国在 2017 年[1]估计，超过 80%的收集污水未经充分处理就被排放。未经处理的污水排放在基础设施、技术资源和有效处理资金不足的发展中国家尤为普遍。例如在墨西哥，2012 年只有 47.5%的市政污水和 28.8%的工业污水得到了处理[2]。为了解决这一问题，墨西哥制定了一项全国性水资源计划，以改善高质量饮用水的获取。该计划旨在通过现代化污水处理厂优先帮助最弱势群体[2]。然而，污水处理厂的高运营成本至今阻碍了进展。采用能够从废物中创造收入的新技术可能提供一种可持续的解决方案。
发展中国家面临污水处理不足及其对健康、经济和环境造成负面影响的重大挑战。尽管关于全球污水产生、收集和处理的代表性数据尚未广泛发布，但联合国在 2017 年[1]估计，超过 80%的收集污水未经充分处理就被排放。未经处理的污水排放在基础设施、技术资源和有效处理资金不足的发展中国家尤为普遍。例如在墨西哥，2012 年只有 47.5%的市政污水和 28.8%的工业污水得到了处理[2]。为了解决这一问题，墨西哥制定了一项全国性水资源计划，以改善高质量饮用水的获取。该计划旨在通过现代化污水处理厂优先帮助最弱势群体[2]。然而，污水处理厂的高运营成本至今阻碍了进展。采用能够从废物中创造收入的新技术可能提供一种可持续的解决方案。
Since the 1950′s, there has been great interest in microalgae wastewater treatment. Microalgae are a diverse group of unicellular microorganisms that efficiently convert inorganic carbon into biomass via photosynthesis whilst consuming nutrients such as nitrates and phosphates. They thrive in a range of environments including wastewater and the resulting microalgal lipids, carbohydrates and proteins provide feedstocks for biodiesel, bioethanol, bioplastic or industrial pigment products [3]. Recent studies have concluded that heterotrophic growth has the potential to overcome some of the major challenges associated with autotrophic cultivation in high rate algal ponds. Unlike autotrophic growth, heterotrophic systems are not limited by light penetration or local weather conditions meaning higher productivities and biomass concentrations can be achieved all year round at large scale. In addition, growth can be carried out in practically any fermenter eliminating the need for large land areas [3]. Although the ability to switch nutritional mode based on substrate availability and light conditions is limited to relatively few microalgae species, Scenedesmus sp. has been successfully grown under heterotrophic conditions, achieving a maximum biomass concentration of 3.46 g/L [4], in six days when supplemented with 10 g/L of glucose. However, these results along with many others reported in literature were obtained from microalgae cultivated under sterile conditions. Under non-sterile heterotrophic conditions, it has been reported that microalgae are unable to compete with other microorganisms such as bacteria and yeast in the culture medium due to their inferior glucose assimilation efficiency [5]. Two microalgae strains, Chlorella protothecoides and Chlamydomonas acidophila were unable to compete with neutral and acidic bacteria, respectively, in wastewater supplemented with a range of glucose concentrations up to 10 g/L [5]. This suggests a sterilisation process such as autoclaving or microfiltration would be required to achieve sufficient sterility for successful heterotrophic microalgae growth in municipal wastewater. As autoclaving is an energy intensive process the high associated costs would render the process economically infeasible.
自 20 世纪 50 年代以来，人们对利用微藻进行废水处理表现出了极大的兴趣。微藻是一类多样化的单细胞微生物，通过光合作用高效地将无机碳转化为生物质，同时消耗硝酸盐和磷酸盐等营养物质。它们能够在包括废水在内的多种环境中生存，而由微藻产生的脂类、碳水化合物和蛋白质可以作为生物柴油、生物乙醇、生物塑料或工业色素产品的原料[3]。 近期研究表明，异养生长有潜力克服与自养培养相关的一些主要挑战。在高效藻类池中，自养生长受限于光线穿透和当地气候条件，而异养系统则不受这些限制，从而可以在全年大规模生产中实现更高的生产率和生物质浓度。此外，异养生长几乎可以在任何发酵罐中进行，无需占用大面积土地[3]。 尽管根据底物供应和光照条件改变营养模式的能力仅限于少数微藻种类，但 Scenedesmus 属在异养条件下已经成功实现生长。在补充 10 g/L 葡萄糖的条件下，其在六天内达到最高生物质浓度 3.46 g/L[4]。然而，这些结果以及文献中报道的许多其他研究结果，均是在无菌条件下培养微藻获得的。在非无菌异养条件下，据报道微藻无法与培养基中的其他微生物（如细菌和酵母）竞争，因为它们的葡萄糖吸收效率较低[5]。 在补充不同浓度（最高 10 g/L）葡萄糖的废水中，两种微藻菌株 Chlorella protothecoides 和 Chlamydomonas acidophila 分别无法与中性和酸性细菌竞争[5]。这表明，为了在市政废水中实现成功的异养微藻生长，需要采用如高压灭菌或微滤这样的灭菌工艺。然而，高压灭菌是一种耗能的过程，其高昂的相关成本使得该过程在经济上不可行。
自 20 世纪 50 年代以来，人们对利用微藻进行废水处理表现出了极大的兴趣。微藻是一类多样化的单细胞微生物，通过光合作用高效地将无机碳转化为生物质，同时消耗硝酸盐和磷酸盐等营养物质。它们能够在包括废水在内的多种环境中生存，而由微藻产生的脂类、碳水化合物和蛋白质可以作为生物柴油、生物乙醇、生物塑料或工业色素产品的原料[3]。 近期研究表明，异养生长有潜力克服与自养培养相关的一些主要挑战。在高效藻类池中，自养生长受限于光线穿透和当地气候条件，而异养系统则不受这些限制，从而可以在全年大规模生产中实现更高的生产率和生物质浓度。此外，异养生长几乎可以在任何发酵罐中进行，无需占用大面积土地[3]。 尽管根据底物供应和光照条件改变营养模式的能力仅限于少数微藻种类，但 Scenedesmus 属在异养条件下已经成功实现生长。在补充 10 g/L 葡萄糖的条件下，其在六天内达到最高生物质浓度 3.46 g/L[4]。然而，这些结果以及文献中报道的许多其他研究结果，均是在无菌条件下培养微藻获得的。在非无菌异养条件下，据报道微藻无法与培养基中的其他微生物（如细菌和酵母）竞争，因为它们的葡萄糖吸收效率较低[5]。 在补充不同浓度（最高 10 g/L）葡萄糖的废水中，两种微藻菌株 Chlorella protothecoides 和 Chlamydomonas acidophila 分别无法与中性和酸性细菌竞争[5]。这表明，为了在市政废水中实现成功的异养微藻生长，需要采用如高压灭菌或微滤这样的灭菌工艺。然而，高压灭菌是一种耗能的过程，其高昂的相关成本使得该过程在经济上不可行。
Whilst yeast have wide spread applications in the brewing, baking and pharmaceutical industries; their application in the wastewater treatment industry has not been fully explored possibly due to the non- sterile conditions. Despite this, phosphorus typically makes up 3–5% of a yeast cell’s dry weight [6] significantly greater than the typical content of 0.87% for microalgae [7]. The nitrogen content of yeast is also higher at 10% [6] compared to around 6% for microalgae [7]. This suggests that yeast may be capable of removing, compared to microalgae, a greater proportion of the nutrients from wastewater. Yeast also present the major advantage of good settling and dewatering properties allowing cost effective removal of biomass. Traditional wastewater treatment plants (WWTP) use activated sludge to remove organic matter. Yeast provide the unique advantage of simultaneous chemical oxygen demand (COD) and nutrient removal. A study by Lim, Kim and Hwang [8] found that yeast were able to reduce the COD of industrial wastewater by 70% in 6.3 h. The dual application of yeast to remove organic matter from municipal wastewater and eliminate the need for additional nutrient removal steps, could minimise operational costs associated with wastewater treatment. Whilst the capability of yeast to dramatically reduce the organic load of industrial wastewaters has been reported, the feasibility of combined COD and nutrient removal by yeast has not been explored. Following wastewater treatment, residual yeast biomass could be utilised for bioethanol production. Bioethanol is the most widely used biofuel owing to its ability to greatly reduce crude oil consumption and atmospheric pollution. Inoculum preparation for corn ethanol production typically requires additional fermentable sugars typically derived from the food crop. Hence, the use of yeast cells produced during wastewater treatment for ethanol biosynthesis would reduce the food crop requirements. Furthermore, the use of treated wastewater effluent would eliminate the need for fresh water for bioethanol production as shown in Ramchandran, Singh, Rajagopalan and Strathmann [9]. Synergically, the economic feasibility of large-scale wastewater treatment would be improved through the generation of a valuable bioethanol product. However, as the capability of yeast to both successfully treat wastewater and produce high quality bioethanol has not been investigated, further research is required.
尽管酵母在酿造、烘焙和制药行业有广泛应用，但由于非无菌条件的存在，其在废水处理行业的应用尚未得到充分探索。尽管如此，磷通常占酵母细胞干重的 3–5% [6]，显著高于微藻的典型含量 0.87% [7]。酵母的氮含量也更高，为 10% [6]，而微藻约为 6% [7]。这表明，与微藻相比，酵母可能能够从废水中去除更多的营养成分。此外，酵母还具有良好的沉降和脱水性能，这使得生物质的去除更加经济。传统的废水处理厂（WWTP）使用活性污泥去除有机物，而酵母在化学需氧量（COD）和养分去除方面具有独特的同步优势。Lim、Kim 和 Hwang [8] 的一项研究发现，酵母能够在 6.3 小时内将工业废水的 COD 降低 70%。将酵母同时用于去除市政废水中的有机物并消除额外的养分去除步骤，可以降低废水处理的运营成本。 尽管已有研究报道了酵母能够显著降低工业废水的有机负荷，但酵母同时去除 COD 和养分的可行性尚未被探索。在废水处理后，残余的酵母生物质可以用于生产生物乙醇。生物乙醇是使用最广泛的生物燃料，因为它能够大幅减少原油消耗和大气污染。玉米乙醇生产的菌种制备通常需要额外的可发酵糖，而这些糖通常来源于粮食作物。因此，将废水处理过程中产生的酵母细胞用于乙醇生物合成，可以减少对粮食作物的需求。此外，如 Ramchandran、Singh、Rajagopalan 和 Strathmann [9] 所示，使用处理后的废水排放物可消除生物乙醇生产对淡水的需求。协同作用下，通过生成高价值的生物乙醇产品，可以提高大规模废水处理的经济可行性。然而，由于酵母同时成功处理废水和生产高质量生物乙醇的能力尚未被研究，因此需要进一步研究。
尽管酵母在酿造、烘焙和制药行业有广泛应用，但由于非无菌条件的存在，其在废水处理行业的应用尚未得到充分探索。尽管如此，磷通常占酵母细胞干重的 3–5% [6]，显著高于微藻的典型含量 0.87% [7]。酵母的氮含量也更高，为 10% [6]，而微藻约为 6% [7]。这表明，与微藻相比，酵母可能能够从废水中去除更多的营养成分。此外，酵母还具有良好的沉降和脱水性能，这使得生物质的去除更加经济。传统的废水处理厂（WWTP）使用活性污泥去除有机物，而酵母在化学需氧量（COD）和养分去除方面具有独特的同步优势。Lim、Kim 和 Hwang [8] 的一项研究发现，酵母能够在 6.3 小时内将工业废水的 COD 降低 70%。将酵母同时用于去除市政废水中的有机物并消除额外的养分去除步骤，可以降低废水处理的运营成本。 尽管已有研究报道了酵母能够显著降低工业废水的有机负荷，但酵母同时去除 COD 和养分的可行性尚未被探索。在废水处理后，残余的酵母生物质可以用于生产生物乙醇。生物乙醇是使用最广泛的生物燃料，因为它能够大幅减少原油消耗和大气污染。玉米乙醇生产的菌种制备通常需要额外的可发酵糖，而这些糖通常来源于粮食作物。因此，将废水处理过程中产生的酵母细胞用于乙醇生物合成，可以减少对粮食作物的需求。此外，如 Ramchandran、Singh、Rajagopalan 和 Strathmann [9] 所示，使用处理后的废水排放物可消除生物乙醇生产对淡水的需求。协同作用下，通过生成高价值的生物乙醇产品，可以提高大规模废水处理的经济可行性。然而，由于酵母同时成功处理废水和生产高质量生物乙醇的能力尚未被研究，因此需要进一步研究。
Interest is emerging in the use of combined symbiotic microalgae and yeast cultivations for improved integrated wastewater treatment and biofuel production [[10], [11], [12]]. Microalgae generate oxygen which is utilised by yeast to assimilate carbon substrates, the yeast subsequently release CO2 promoting microalgal photosynthesis. As the negative charge of microalgal cells prevents self-flocculation, a costly and/or energy intensive flocculation step is required to recover valuable biomass. Fungal species which possess effective flocculation properties have been found to effectively trap microalgae cells aiding biomass recovery [13]. Iasimone, Zuccaro, D'Oriano, Franci, Galdiero, Pirozzi, De Felice and Pirozzi [11], found a mixture of an oleaginous yeast strain, Lipomyces starkeyi, and the native wastewater microalga, Scenedesmus sp. and Chlorella sp. reduced the lag phase associated with microalgae growth by three days compared to the microalgae alone. This was attributed to CO2 enrichment resulting from yeast respiration. Alkalinisation during microalgae growth increased the pH of the culture medium to 10.9 which had beneficial antibacterial and antifungal effects leading to a predominantly microalgal culture after 14 days of cultivation. Although biomass and lipid productivity increased, the final biomass and lipid concentrations were around 300 and 45 mg/L, respectively. As a result of the aforementioned high costs associated with the running of raceway ponds, such yields are likely to be insufficient for sustainable combined wastewater treatment and biodiesel production. The cultivation of a mixture of the microalgae and yeast strains, Chlorella vulgaris and Yarrowia lipolytica by Qin, Wei, Wang and Alam [12], achieved significantly higher biomass and lipid yields of up to 1.53 g/L and 187 mg/L, respectively. However, synthetic wastewater and an energy intensive sterilisation process were employed.
近年来，人们开始关注利用共生微藻和酵母联合培养来改善综合废水处理和生物燃料生产的研究[[10], [11], [12]]。微藻产生的氧气可被酵母利用来同化碳基底物，而酵母随后释放的 CO₂促进了微藻的光合作用。由于微藻细胞的负电荷阻碍其自絮凝，因此需要耗费高成本和/或高能耗的絮凝步骤来回收有价值的生物质。研究发现，具有有效絮凝特性的真菌可以有效捕获微藻细胞，从而有助于生物质的回收[13]。Iasimone、Zuccaro、D'Oriano、Franci、Galdiero、Pirozzi、De Felice 和 Pirozzi [11]发现，一种富含油脂的酵母菌株 Lipomyces starkeyi 与本地废水微藻 Scenedesmus sp.和 Chlorella sp.的混合培养相比单独培养微藻，减少了微藻生长的滞后期三天。这归因于酵母呼吸作用产生的 CO₂浓度增加。微藻生长过程中发生的碱化作用将培养基的 pH 值提高到 10.9，这对抗菌和抗真菌具有积极作用，使得在培养 14 天后形成以微藻为主的培养物。尽管生物质和脂质生产力有所提高，但最终的生物质和脂质浓度分别约为 300 mg/L 和 45 mg/L。由于上述运行跑道池的高成本，这样的产量可能不足以支持可持续的综合废水处理和生物柴油生产。Qin、Wei、Wang 和 Alam [12]通过培养微藻 Chlorella vulgaris 和酵母 Yarrowia lipolytica 的混合物，显著提高了生物质和脂质产量，分别达到了 1.53 g/L 和 187 mg/L。然而，该研究中使用了合成废水并采用了高能耗的灭菌过程。
近年来，人们开始关注利用共生微藻和酵母联合培养来改善综合废水处理和生物燃料生产的研究[[10], [11], [12]]。微藻产生的氧气可被酵母利用来同化碳基底物，而酵母随后释放的 CO₂促进了微藻的光合作用。由于微藻细胞的负电荷阻碍其自絮凝，因此需要耗费高成本和/或高能耗的絮凝步骤来回收有价值的生物质。研究发现，具有有效絮凝特性的真菌可以有效捕获微藻细胞，从而有助于生物质的回收[13]。Iasimone、Zuccaro、D'Oriano、Franci、Galdiero、Pirozzi、De Felice 和 Pirozzi [11]发现，一种富含油脂的酵母菌株 Lipomyces starkeyi 与本地废水微藻 Scenedesmus sp.和 Chlorella sp.的混合培养相比单独培养微藻，减少了微藻生长的滞后期三天。这归因于酵母呼吸作用产生的 CO₂浓度增加。微藻生长过程中发生的碱化作用将培养基的 pH 值提高到 10.9，这对抗菌和抗真菌具有积极作用，使得在培养 14 天后形成以微藻为主的培养物。尽管生物质和脂质生产力有所提高，但最终的生物质和脂质浓度分别约为 300 mg/L 和 45 mg/L。由于上述运行跑道池的高成本，这样的产量可能不足以支持可持续的综合废水处理和生物柴油生产。Qin、Wei、Wang 和 Alam [12]通过培养微藻 Chlorella vulgaris 和酵母 Yarrowia lipolytica 的混合物，显著提高了生物质和脂质产量，分别达到了 1.53 g/L 和 187 mg/L。然而，该研究中使用了合成废水并采用了高能耗的灭菌过程。
The aim of this study was to investigate, for the first time, the feasibility of novel non-sterile integrated municipal wastewater treatment combined with bioethanol production for improved sustainability. Both, microalgae (mostly Scenedesmus sp.) and a consortium of yeast isolated from municipal wastewater were grown under non-sterile heterotrophic conditions. The effectivity of co-cultivation or single strain cultivation was assessed in terms of strain growth, COD and nutrient removal during wastewater treatment.
本研究的目的是首次研究一种新型非无菌一体化市政废水处理与生物乙醇生产相结合的可行性，以提高可持续性。在非无菌异养条件下，培养了微藻（主要为绿球藻属 Scenedesmus sp.）和从市政废水中分离出的酵母菌群。通过废水处理中菌株的生长、化学需氧量（COD）和营养物去除效果，评估了联合培养与单菌株培养的有效性。
本研究的目的是首次研究一种新型非无菌一体化市政废水处理与生物乙醇生产相结合的可行性，以提高可持续性。在非无菌异养条件下，培养了微藻（主要为绿球藻属 Scenedesmus sp.）和从市政废水中分离出的酵母菌群。通过废水处理中菌株的生长、化学需氧量（COD）和营养物去除效果，评估了联合培养与单菌株培养的有效性。
2. Materials and methods 2. 材料与方法
Several wastewater treatment processes were studied using wild yeast and microalgae. Cultures were tested under non-sterile heterotrophic conditions to evaluate wastewater treatment and the simultaneous production of high value biomass. Three systems were investigated sequentially in batch bioreactors (Table 2): 1) Cultures with a predominance of microalgae; 2) Co-cultures of microalgae and wild yeast; 3) Cultures with a predominance of wild yeast. Nutrient removal efficiency and biomass accumulation were monitored for each of the aforementioned culture systems over a period of eight days.
使用野生酵母和微藻研究了几种废水处理工艺。在非无菌异养条件下对培养物进行了测试，以评估废水处理效果并同时生产高价值生物质。在批量生物反应器中依次研究了三种系统（表 2）：1）以微藻为主的培养；2）微藻与野生酵母的共培养；3）以野生酵母为主的培养。在八天的时间内，监测了上述每种培养系统的营养去除效率和生物质积累情况。
使用野生酵母和微藻研究了几种废水处理工艺。在非无菌异养条件下对培养物进行了测试，以评估废水处理效果并同时生产高价值生物质。在批量生物反应器中依次研究了三种系统（表 2）：1）以微藻为主的培养；2）微藻与野生酵母的共培养；3）以野生酵母为主的培养。在八天的时间内，监测了上述每种培养系统的营养去除效率和生物质积累情况。
2.1. Wastewater preparation and characterisation
2.1. 废水制备与特性分析
Municipal wastewater provided the culture medium in all bioreactors, this was collected from the effluent of a treatment plant located on the central campus of the Universidad Nacional Autónoma de Mexico. Prior to collection, the wastewater underwent primary and secondary treatment. This involved two sequential bio-towers, in the first organic matter was removed whilst in the second partial nitrification was performed to reduce the organic load.
**翻译为简体中文：** 所有生物反应器中的培养基均由市政废水提供，这些废水取自墨西哥国立自治大学中央校区一座处理厂的出水。在采集之前，废水经过了一级和二级处理。这包括两个连续的生物塔，在第一个生物塔中去除了有机物，而在第二个生物塔中进行了部分硝化以减少有机负载。
**翻译为简体中文：** 所有生物反应器中的培养基均由市政废水提供，这些废水取自墨西哥国立自治大学中央校区一座处理厂的出水。在采集之前，废水经过了一级和二级处理。这包括两个连续的生物塔，在第一个生物塔中去除了有机物，而在第二个生物塔中进行了部分硝化以减少有机负载。
Culture medium preparation involved the filtration of wastewater samples on site to remove large suspended solids using a filter with an 8 μm pore size. The filtered samples were then stored at 4 °C in 20 L high density polyethylene (HDPE) containers until use. As the initial concentration of nutrients varied depending on collection time, samples of the filtered wastewater were characterised according to the following standard methods.
培养基的制备是在现场对废水样本进行过滤，以去除较大的悬浮固体，使用孔径为 8 μm 的过滤器。过滤后的样本存储在容量为 20 L 的高密度聚乙烯（HDPE）容器中，并在 4°C 下保存直至使用。由于初始养分浓度因采集时间而异，因此对过滤后的废水样本按照以下标准方法进行了表征。
培养基的制备是在现场对废水样本进行过滤，以去除较大的悬浮固体，使用孔径为 8 μm 的过滤器。过滤后的样本存储在容量为 20 L 的高密度聚乙烯（HDPE）容器中，并在 4°C 下保存直至使用。由于初始养分浓度因采集时间而异，因此对过滤后的废水样本按照以下标准方法进行了表征。
Nitrate and orthophosphate concentrations were measured using the HACH® cadmium reduction and amino acid methods, respectively [14], whilst COD was monitored using the HACH® reactor digestion method [14]. To avoid interference, due to the colorimetric nature of the method, each sample was filtered using a Whatman® GF/A glass fibre filter with a 1.6 μm pore size prior to the addition of reagents and measurement using a HACH® DR3900 Spectrophotometer. Total ammonia nitrogen (TAN) concentration was determined using a modified Total Kjeldahl Nitrogen (TKN) method [15] using a BÜCHI B-324 Distillation Unit. In aqueous solution, ammonia exists as an equilibrium of its two principal forms, unionised ammonia (NH3) and ammonium ions (NH4+). The relative concentrations of these depend on temperature and pH. TAN was given by the sum of the NH3 and NH4+ concentrations. The standard TKN method quantifies both inorganic and organic nitrogen. Both microalgae and yeast are rich in organic nitrogen which could lead to false positive results. It was therefore necessary to omit the organic nitrogen digestion step to facilitate measurement of the TAN concentration.
硝酸盐和正磷酸盐浓度分别使用 HACH®镉还原法和氨基酸法测量[14]，而化学需氧量（COD）通过 HACH®反应消解法监测[14]。为了避免因比色法的特性引起干扰，每个样本在加入试剂和使用 HACH® DR3900 分光光度计测量之前，使用孔径为 1.6 μm 的 Whatman® GF/A 玻璃纤维滤纸进行过滤。总氨氮（TAN）浓度使用改进的总凯氏氮（TKN）方法[15]，通过 BÜCHI B-324 蒸馏设备测定。在水溶液中，氨以两种主要形式存在的平衡状态：非离子氨（NH₃）和铵离子（NH₄⁺）。它们的相对浓度取决于温度和 pH 值。TAN 由 NH₃和 NH₄⁺浓度之和表示。标准的 TKN 方法可同时量化无机氮和有机氮。由于微藻和酵母富含有机氮，这可能导致假阳性结果，因此有必要省略有机氮消化步骤，以便测量 TAN 浓度。
硝酸盐和正磷酸盐浓度分别使用 HACH®镉还原法和氨基酸法测量[14]，而化学需氧量（COD）通过 HACH®反应消解法监测[14]。为了避免因比色法的特性引起干扰，每个样本在加入试剂和使用 HACH® DR3900 分光光度计测量之前，使用孔径为 1.6 μm 的 Whatman® GF/A 玻璃纤维滤纸进行过滤。总氨氮（TAN）浓度使用改进的总凯氏氮（TKN）方法[15]，通过 BÜCHI B-324 蒸馏设备测定。在水溶液中，氨以两种主要形式存在的平衡状态：非离子氨（NH₃）和铵离子（NH₄⁺）。它们的相对浓度取决于温度和 pH 值。TAN 由 NH₃和 NH₄⁺浓度之和表示。标准的 TKN 方法可同时量化无机氮和有机氮。由于微藻和酵母富含有机氮，这可能导致假阳性结果，因此有必要省略有机氮消化步骤，以便测量 TAN 浓度。
Finally, the carbohydrate concentration was monitored using the phenol-sulphuric acid method [16]. A HACH® DR3900 Spectrophotometer was used to measure absorbance at 490 nm; and concentrations were determined from a calibration curve produced using known concentrations of glucose. Filtration of the sample using a Whatman® GF/A glass fibre filter with a 1.6 μm pore size was also performed prior to reagent addition to remove cells containing internal carbohydrates and ensure the concentration corresponded to that of the culture medium. Summary of wastewater characteristics obtained from four wastewater samples collected over an eight week period are shown in Table 1.
最后，使用苯酚-硫酸法监测了碳水化合物浓度 [16]。使用 HACH® DR3900 分光光度计在 490 nm 处测量吸光度，并通过使用已知浓度的葡萄糖制作的校准曲线确定浓度。在加入试剂之前，样品通过 Whatman® GF/A 玻璃纤维滤膜（孔径为 1.6 μm）进行过滤，以去除含有内部碳水化合物的细胞，并确保测得的浓度与培养基中的浓度一致。表 1 显示了在八周内收集的四个污水样本的污水特性摘要。
最后，使用苯酚-硫酸法监测了碳水化合物浓度 [16]。使用 HACH® DR3900 分光光度计在 490 nm 处测量吸光度，并通过使用已知浓度的葡萄糖制作的校准曲线确定浓度。在加入试剂之前，样品通过 Whatman® GF/A 玻璃纤维滤膜（孔径为 1.6 μm）进行过滤，以去除含有内部碳水化合物的细胞，并确保测得的浓度与培养基中的浓度一致。表 1 显示了在八周内收集的四个污水样本的污水特性摘要。
Table 1. Characterisation of municipal wastewater culture medium. Values shown are mean values ± standard deviation obtained for four samples collected over an eight week period.
表 1. 市政污水培养基的特性。所示数值为在八周内收集的四个样本的平均值 ± 标准偏差。
| Parameter 参数 | Value 价值 |
|---|---|
| Nitrate Concentration (mg/L) 硝酸盐浓度（毫克/升） | 15.0 ± 4.9 |
| Orthophosphate Concentration (mg/L) 正磷酸盐浓度（毫克/升） | 67.5 ± 19.5 |
| TAN Concentration (mg/L) 氨氮浓度 (毫克/升) | 133.1 ± 13.9 |
| Chemical Oxygen Demand (COD) (mg/L) 化学需氧量 (COD) (毫克/升) | 95 ± 24.7 |
| Total Suspended Solids (TSS) (mg/L) 悬浮固体总量 (TSS) (毫克/升) | 47.9 ± 35.8 |
| pH 酸碱度 | 7.5 |
| Carbohydrates (mg/L) 碳水化合物 (毫克/升) | 5.7 ± 3.9 |
Table 2. Cultivation systems evaluated for wastewater treatment.
表 2. 用于废水处理的培养系统评估。
| Number (#) 号码 (#) | Cultivation Name 修炼名称 | Starting inoculum (concentration) 起始接种物（浓度） | Glucose (g/L) 葡萄糖 (g/L) | Final pH 最终 pH 值 | Time (days) 时间 (天) | # B.E. # 工程学士（B.E.） |
|---|---|---|---|---|---|---|
| 1 | Microalgae, Batch 1 微藻，第 1 批 | Microalgae plus wastewater microbes (total of 0.2 ± 0.02 g/L) 微藻加废水微生物（总量为 0.2 ± 0.02 g/L） | 0-20 | 7–8 | 8 | 2 |
| 2 | Microalgae/Yeast, 微藻/酵母 Batch 1 批次 1 | Microalgae from #1 (0.4 ± 0.03 g/L) plus wastewater with yeast (0.03 g/L) giving a total of 0.2 g/L (94 wt% microalgae, 6 wt% yeast) #1 的微藻（0.4 ± 0.03 g/L）加上含酵母的废水（0.03 g/L），总计为 0.2 g/L（微藻占 94 wt%，酵母占 6 wt%）。 | 10,20 | 7 | 7 | 3 |
| 3 | Yeast, Batch 1 酵母，第 1 批次 | Co-culture from #2 (1.9 g/L) plus wastewater giving a total of 0.2 g/L #2 的共培养物（1.9 g/L）加上废水，总计为 0.2 g/L。 | 10 | 4–5 | 8 | 3 |
| 4 | Yeast, Batch 2 酵母，第 2 批 | Culture from #3 (3.7 g/L) plus wastewater giving a total of 0.2 g/L 来自#3 (3.7 g/L)的培养液与废水混合，总浓度为 0.2 g/L | 10 | 4–5 | 8 | 3 |
| 5 | Yeast, Batch 3 酵母，第 3 批 | Culture from #4 (4.2 g/L) plus wastewater giving a total of 0.2 g/L 从#4 培养物（4.2 g/L）和废水混合，总浓度为 0.2 g/L。 | 10 | 4–5 | 2 | 3 |
#BE: Number of biological experimental replicates run in parallel. All cultivations had controls run in parallel.
#BE：平行运行的生物实验重复次数。所有培养实验均有平行对照组。
#BE：平行运行的生物实验重复次数。所有培养实验均有平行对照组。
2.2. Experimental setup and analytical procedure
2.2 实验设置和分析程序
Wastewater treatment performance in each of the culture systems (Table 2) was studied in 5 L batch polyethylene terephthalate (PET) bioreactors (L:14 cm; W:12 cm; H: 25 cm) wrapped in aluminium foil to maintain heterotrophic conditions with a working volume of 60%. Air was supplied continuously using an ELITE 802 dual outlet air pump at a rate of 0.5 VVM. Initial trials found that an additional carbon source was required to sustain growth. Glucose was selected to increase the low COD of municipal wastewater (Table 1) as it is the preferred sugar for the microorganisms used in this study. Several concentrations between 0 and 10 g/L were initially investigated and 10 g/L was ultimately chosen for subsequent experiments (Table 2). Duplicate measurements of nitrate, orthophosphate and, TAN were taken for all experiments. Biomass concentration was measured as total suspended solids (TSS) following the APHA 2540 D standard method [17], cultures were agitated thoroughly prior to taking the measurement to ensure heterogeneity and break up any microbial flocs. Microscope images were taken using a Zeiss Axio Lab A1 microscope with a 0.5x camera adapter. Objective values of 63 and 100X, equivalent to total zoom values of 31.5 and 50X, respectively, were used.
在每种培养系统中的废水处理性能（表 2）研究是在 5 升批量聚对苯二甲酸乙二醇酯（PET）生物反应器中进行的（长：14 厘米；宽：12 厘米；高：25 厘米），外包铝箔以维持异养条件，工作体积为 60%。通过 ELITE 802 双出口空气泵以 0.5 VVM 的速率连续供气。初步试验发现需要额外的碳源来维持生长。选择葡萄糖来提高市政废水的低 COD（表 1），因为它是本研究中所用微生物的优选糖类。最初研究了 0 至 10 g/L 之间的多种浓度，最终选择 10 g/L 用于后续实验（表 2）。所有实验均对硝酸盐、正磷酸盐和 TAN 进行了重复测量。采用 APHA 2540 D 标准方法[17]通过总悬浮固体（TSS）测量生物量浓度，测量前对培养物进行了充分搅拌，以确保均匀性并打散微生物絮体。显微镜图像使用蔡司 Axio Lab A1 显微镜拍摄，配备 0.5 倍相机适配器，使用 63 倍和 100 倍物镜，分别相当于总放大倍数 31.5 倍和 50 倍。
在每种培养系统中的废水处理性能（表 2）研究是在 5 升批量聚对苯二甲酸乙二醇酯（PET）生物反应器中进行的（长：14 厘米；宽：12 厘米；高：25 厘米），外包铝箔以维持异养条件，工作体积为 60%。通过 ELITE 802 双出口空气泵以 0.5 VVM 的速率连续供气。初步试验发现需要额外的碳源来维持生长。选择葡萄糖来提高市政废水的低 COD（表 1），因为它是本研究中所用微生物的优选糖类。最初研究了 0 至 10 g/L 之间的多种浓度，最终选择 10 g/L 用于后续实验（表 2）。所有实验均对硝酸盐、正磷酸盐和 TAN 进行了重复测量。采用 APHA 2540 D 标准方法[17]通过总悬浮固体（TSS）测量生物量浓度，测量前对培养物进行了充分搅拌，以确保均匀性并打散微生物絮体。显微镜图像使用蔡司 Axio Lab A1 显微镜拍摄，配备 0.5 倍相机适配器，使用 63 倍和 100 倍物镜，分别相当于总放大倍数 31.5 倍和 50 倍。
2.2.1. Microalgae culture preparation
2.2.1 微藻培养准备
The microalgae used to inoculate the heterotrophic bioreactors were pre-adapted to wastewater as per [18] using Scenedesmus obliquus (Turpin) Kützing as provided by Instituto Politécnico Nacional, Mexico City, México. Bioreactors were run as per Table 2, #1 including control cultures containing just wastewater, without microalgae or glucose. The inoculum fluid was mixed with filtered wastewater (1:2 v/v) giving each reactor an initial biomass concentration of 0.2 ± 0.02 g/L. Following inoculation, the cultures contained 15.1 ± 1.3 mg/L nitrates, 81.0 ± 0.7 mg/L TAN and 44.2 ± 1.2 mg/L orthophosphates. The pH of each culture was also measured on each of the eight days and volumes of 6 N NaOH added, as required to maintain the pH of the culture medium within the optimal range for microalgae between 7 and 8. The results of this first investigation indicated that a concentration of 10 g/L glucose was optimal for biomass production.
用于接种异养生物反应器的微藻是根据文献[18]使用斜生栅藻（*Scenedesmus obliquus* (Turpin) Kützing），由墨西哥城的国立理工学院提供，并预先适应了废水。生物反应器的运行条件如表 2 所示，包括对照组培养物（仅含废水，无微藻或葡萄糖）。接种液与过滤后的废水按 1:2（体积比）混合，使每个反应器的初始生物量浓度达到 0.2 ± 0.02 g/L。接种后，培养液中含有 15.1 ± 1.3 mg/L 硝酸盐、81.0 ± 0.7 mg/L 总氨氮（TAN）和 44.2 ± 1.2 mg/L 正磷酸盐。在为期 8 天的培养中，每天测量培养液的 pH 值，并根据需要添加 6 N NaOH 以维持培养基的 pH 值在微藻最适范围 7 至 8 之间。首次研究结果表明，葡萄糖浓度为 10 g/L 时，生物量生产效果最佳。
用于接种异养生物反应器的微藻是根据文献[18]使用斜生栅藻（*Scenedesmus obliquus* (Turpin) Kützing），由墨西哥城的国立理工学院提供，并预先适应了废水。生物反应器的运行条件如表 2 所示，包括对照组培养物（仅含废水，无微藻或葡萄糖）。接种液与过滤后的废水按 1:2（体积比）混合，使每个反应器的初始生物量浓度达到 0.2 ± 0.02 g/L。接种后，培养液中含有 15.1 ± 1.3 mg/L 硝酸盐、81.0 ± 0.7 mg/L 总氨氮（TAN）和 44.2 ± 1.2 mg/L 正磷酸盐。在为期 8 天的培养中，每天测量培养液的 pH 值，并根据需要添加 6 N NaOH 以维持培养基的 pH 值在微藻最适范围 7 至 8 之间。首次研究结果表明，葡萄糖浓度为 10 g/L 时，生物量生产效果最佳。
2.2.2. Microalgae and yeast co-culture preparation
2.2.2. 微藻与酵母共培养制备
Concentrated microalgae cultures were used to prepare the inoculum for the microalgae and yeast co-cultures (Table 2, #2) and additional control reactors containing only wastewater supplemented with 10 g/L of glucose. The cultures resulting from microalgae experiments were mixed producing a stock culture with 0.4 ± 0.03 g/L of biomass, which was then added to wastewater containing 0.03 g/L of yeast (1:1 v/v). The resulting cultures had an initial total biomass concentration of 0.2 g/L (94 wt% microalgae, 6 wt% yeast) along with 66.2 ± 4.9 mg/L nitrates, 76.2 ± 0.3 mg/L TAN and 78.5 ± 1.0 mg/L orthophosphates. The pH of each culture was measured each day and volumes of 6 N NaOH added, as required, to maintain neutral pH. Relative microalgae growth was assessed at the end of the fermentations by microscopy. Cultures were mixed thoroughly to ensure heterogeneity before a series of images were taken for samples of triplicate bioreactors. The mean number of microalgae cells was calculated for each of the glucose concentrations studied.
浓缩的微藻培养物被用来制备微藻和酵母的共培养接种物（表 2，#2），以及仅含有废水和 10 g/L 葡萄糖的额外对照反应器。微藻实验产生的培养物被混合，制备了一个含有 0.4 ± 0.03 g/L 生物量的储备培养物，然后将其加入含有 0.03 g/L 酵母的废水中（1:1 v/v）。最终培养物的初始总生物量浓度为 0.2 g/L（94 wt%微藻，6 wt%酵母），同时含有 66.2 ± 4.9 mg/L 硝酸盐、76.2 ± 0.3 mg/L 氨氮（TAN）和 78.5 ± 1.0 mg/L 正磷酸盐。每种培养物的 pH 值每天都被测量，并根据需要添加 6 N NaOH 以维持中性 pH 值。通过显微镜评估发酵结束时的相对微藻生长情况。为了确保样品的均匀性，在对三重复反应器的样品进行图像拍摄之前，培养物被充分混合。对于研究的每种葡萄糖浓度，计算得到的微藻细胞的平均数量。
浓缩的微藻培养物被用来制备微藻和酵母的共培养接种物（表 2，#2），以及仅含有废水和 10 g/L 葡萄糖的额外对照反应器。微藻实验产生的培养物被混合，制备了一个含有 0.4 ± 0.03 g/L 生物量的储备培养物，然后将其加入含有 0.03 g/L 酵母的废水中（1:1 v/v）。最终培养物的初始总生物量浓度为 0.2 g/L（94 wt%微藻，6 wt%酵母），同时含有 66.2 ± 4.9 mg/L 硝酸盐、76.2 ± 0.3 mg/L 氨氮（TAN）和 78.5 ± 1.0 mg/L 正磷酸盐。每种培养物的 pH 值每天都被测量，并根据需要添加 6 N NaOH 以维持中性 pH 值。通过显微镜评估发酵结束时的相对微藻生长情况。为了确保样品的均匀性，在对三重复反应器的样品进行图像拍摄之前，培养物被充分混合。对于研究的每种葡萄糖浓度，计算得到的微藻细胞的平均数量。
2.2.3. Yeast culture preparation
2.2.3 酵母培养制备
In the third system, biomass resulting from replicate co-cultures was mixed, giving a stock culture with a TSS concentration of 3.5 g/L. This was then added to wastewater in a 1:14 v/v inoculum:wastewater ratio. Following inoculation each reactor contained 16.2 ± 1.1 mg/L of nitrates, 44.5 ± 0.6 mg/L of orthophosphates, 141.2 ± 1.2 mg/L of TAN, 10,016 ± 1.9 mg/L of carbohydrates and 10,821 ± 5.3 mg/L of COD. Each tested batch was run as per Table 2, #3-5, plus additional control bioreactors containing wastewater supplemented with 10 g/L of glucose. After the first batch, ethanol concentration was monitored in subsequent batches and this was achieved by distillation using a BÜCHI B-324 Distillation Unit following the manufacturer’s instructions [19].
在第三个系统中，将复制共培养生成的生物质混合，制备了总悬浮固体（TSS）浓度为 3.5 g/L 的储备培养物。然后以 1:14 的接种物与废水体积比加入废水中。接种后，每个反应器中含有 16.2 ± 1.1 mg/L 的硝酸盐、44.5 ± 0.6 mg/L 的正磷酸盐、141.2 ± 1.2 mg/L 的总氨氮（TAN）、10,016 ± 1.9 mg/L 的碳水化合物以及 10,821 ± 5.3 mg/L 的化学需氧量（COD）。每个测试批次按照表 2 中的#3-5 运行，并增加了含有 10 g/L 葡萄糖的废水作为对照生物反应器。在第一个批次后，随后批次中通过使用 BÜCHI B-324 蒸馏装置并遵循制造商说明[19]的方法监测乙醇浓度。
在第三个系统中，将复制共培养生成的生物质混合，制备了总悬浮固体（TSS）浓度为 3.5 g/L 的储备培养物。然后以 1:14 的接种物与废水体积比加入废水中。接种后，每个反应器中含有 16.2 ± 1.1 mg/L 的硝酸盐、44.5 ± 0.6 mg/L 的正磷酸盐、141.2 ± 1.2 mg/L 的总氨氮（TAN）、10,016 ± 1.9 mg/L 的碳水化合物以及 10,821 ± 5.3 mg/L 的化学需氧量（COD）。每个测试批次按照表 2 中的#3-5 运行，并增加了含有 10 g/L 葡萄糖的废水作为对照生物反应器。在第一个批次后，随后批次中通过使用 BÜCHI B-324 蒸馏装置并遵循制造商说明[19]的方法监测乙醇浓度。
2.3. Statistical analysis
2.3. 统计分析
Statistical analysis was performed using Minitab 17 statistical software and MATLAB R2017a. T-tests were performed in Minitab® using a significance level of 0.05 to analyse and compare results from different parallel batches. The null hypothesis considered that there was no significant difference between the cultures, hence if p ≤ 0.05 the null hypothesis was rejected. The wastewater composition varied depending on time of collection. A test for equal variance was therefore performed using Minitab to assess the repeatability of yeast growth between batches of replicates. Linear regression was also performed using MATLAB by plotting results of the first batch against those of the second batch. The resulting R2 values for biomass, orthophosphate, carbohydrate and TAN were 0.9535, 0.9842, 0.9979 and 0.987 respectively indicating a high level of repeatability between batches.
统计分析使用 Minitab 17 统计软件和 MATLAB R2017a 进行。在 Minitab® 中使用显著性水平为 0.05 的 t 检验分析和比较不同平行批次的结果。零假设认为各培养物之间没有显著差异，因此如果 p ≤ 0.05，则拒绝零假设。废水组成因采集时间的不同而有所变化。因此，使用 Minitab 进行了等方差检验，以评估酵母在不同重复批次之间的生长重复性。还使用 MATLAB 通过将第一批结果与第二批结果作图进行线性回归分析。生物量、正磷酸盐、碳水化合物和 TAN 的 R 值分别为 0.9535、0.9842、0.9979 和 0.987，表明批次之间具有高度的重复性。
统计分析使用 Minitab 17 统计软件和 MATLAB R2017a 进行。在 Minitab® 中使用显著性水平为 0.05 的 t 检验分析和比较不同平行批次的结果。零假设认为各培养物之间没有显著差异，因此如果 p ≤ 0.05，则拒绝零假设。废水组成因采集时间的不同而有所变化。因此，使用 Minitab 进行了等方差检验，以评估酵母在不同重复批次之间的生长重复性。还使用 MATLAB 通过将第一批结果与第二批结果作图进行线性回归分析。生物量、正磷酸盐、碳水化合物和 TAN 的 R 值分别为 0.9535、0.9842、0.9979 和 0.987，表明批次之间具有高度的重复性。
2.4. Yeast identification: DNA extraction, sequencing methods and analysis
2.4. 酵母鉴定：DNA 提取、测序方法与分析
Dried samples of microalgae and yeast co-cultures underwent DNA extraction. 200 mg of dried sample was weighed into a Lysing Matirx E tube and hydrated with 300 μL of molecular-grade H2O. DNA was then extracted using the FastDNA Spin Kit for Soil (MP Biomedicals, USA) as per the manufacturer’s instructions. Extracted DNA was subsequently diluted 1:10 with molecular-grade H2O in order to dilute out any PCR-inhibiting substances that were co-extracted. Next, diluted template DNA was quantified using a Qubit 3.0 fluorometer using the dsDNA HS Assay Kit (Life Technologies, USA) according to the manufacturer’s instructions. Diluted template DNA then underwent PCR targeting the fungal ITS1 region, using the primers ITS1f (5` – CTTGGTCATTTAGAGGAAGTAA) [20] and ITS2 (5` – GCTGCGTTCTTCATCGATGC) [21] as per the EMP Protocol [20]. The PCR involved a 10 μL reaction comprised of: 9 μL of MegaMix Blue (Microzone, UK), which contained taq polymerase, dNTPs, MgCl2–, buffer, and agarose loading dye; 0.25 μL of each primer (100 pM concentration) and; 0.5 μL of 1:10 diluted template DNA. PCR conditions were as follows: Initial denaturation at 94 °C for 1 min, followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 68 °C for 30 s, and a final elongation step of 68 °C for 10 min. PCR amplification was performed on a Techne 512 thermocycler. Procedural positive was DNA from Trichoderma reesei (DSMZ no. 768) and procedural negative was molecular grade water. Following PCR, amplicon product and fragment size checks were performed by agarose gel electrophoresis. The agarose gel was comprised of 1 g of agarose (UltraPure, Invitogen, USA), 99 mL of 1 x TAE (tris-acetate-EDTA) buffer and 1.6 μL of ethidium bromide (Bio-Rad, USA). As the MegaMix Blue reagent contained loading dye it was not necessary to add any additional dye. Marker lanes were loaded with 2.5 μL of HyperLadder 50bp (BioLine, UK), which contained DNA fragments of varying sizes, ranging from 50 bp to 2.1 kbp, to determine PCR amplicon fragment size. Electrophoresis was performed for 45 min at 100 V. Gels were visualised using a UV MultiDoc-It™ Imaging System (UVP, USA).
微藻和酵母共培养物的干燥样品进行了 DNA 提取。称取 200 mg 的干燥样品置于 Lysing Matrix E 管中，并加入 300 μL 分子级 H₂O 进行水化。随后，按照制造商说明使用 FastDNA Spin Kit for Soil（MP Biomedicals，美国）提取 DNA。提取的 DNA 用分子级 H₂O 按 1:10 稀释，以稀释掉共提取的可能抑制 PCR 的物质。接着，使用 Qubit 3.0 荧光计和 dsDNA HS Assay Kit（Life Technologies，美国）按照制造商说明对稀释后的模板 DNA 进行定量。之后，稀释后的模板 DNA 进行靶向真菌 ITS1 区的 PCR，使用引物 ITS1f（5` – CTTGGTCATTTAGAGGAAGTAA）[20]和 ITS2（5` – GCTGCGTTCTTCATCGATGC）[21]，按照 EMP Protocol [20]操作。 PCR 反应体系为 10 μL，包括：9 μL 含 Taq 聚合酶、dNTPs、MgCl₂、缓冲液和琼脂糖上样染料的 MegaMix Blue（Microzone，英国）；0.25 μL（100 pM 浓度）的每种引物；0.5 μL 按 1:10 稀释的模板 DNA。PCR 条件如下：初始变性 94 °C 1 分钟；随后进行 35 个循环：94 °C 30 秒，52 °C 30 秒，68 °C 30 秒；最后延伸 68 °C 10 分钟。PCR 扩增在 Techne 512 热循环仪上进行。阳性对照为来自**Trichoderma reesei**（DSMZ 编号 768）的 DNA，阴性对照为分子级水。 PCR 后，通过琼脂糖凝胶电泳对扩增子产物和片段大小进行检查。琼脂糖凝胶配方为：1 g 琼脂糖（UltraPure，Invitrogen，美国）、99 mL 1 x TAE（Tris-乙酸-EDTA）缓冲液和 1.6 μL 溴化乙锭（Bio-Rad，美国）。由于 MegaMix Blue 试剂中已含有上样染料，因此无需额外添加。标记物通道加载 2.5 μL HyperLadder 50 bp（BioLine，英国），其包含从 50 bp 到 2.1 kbp 范围内不同大小的 DNA 片段，用于确定 PCR 扩增子片段大小。电泳在 100 V 条件下运行 45 分钟。凝胶通过 UV MultiDoc-It™成像系统（UVP，美国）进行紫外线观察。
微藻和酵母共培养物的干燥样品进行了 DNA 提取。称取 200 mg 的干燥样品置于 Lysing Matrix E 管中，并加入 300 μL 分子级 H₂O 进行水化。随后，按照制造商说明使用 FastDNA Spin Kit for Soil（MP Biomedicals，美国）提取 DNA。提取的 DNA 用分子级 H₂O 按 1:10 稀释，以稀释掉共提取的可能抑制 PCR 的物质。接着，使用 Qubit 3.0 荧光计和 dsDNA HS Assay Kit（Life Technologies，美国）按照制造商说明对稀释后的模板 DNA 进行定量。之后，稀释后的模板 DNA 进行靶向真菌 ITS1 区的 PCR，使用引物 ITS1f（5` – CTTGGTCATTTAGAGGAAGTAA）[20]和 ITS2（5` – GCTGCGTTCTTCATCGATGC）[21]，按照 EMP Protocol [20]操作。 PCR 反应体系为 10 μL，包括：9 μL 含 Taq 聚合酶、dNTPs、MgCl₂、缓冲液和琼脂糖上样染料的 MegaMix Blue（Microzone，英国）；0.25 μL（100 pM 浓度）的每种引物；0.5 μL 按 1:10 稀释的模板 DNA。PCR 条件如下：初始变性 94 °C 1 分钟；随后进行 35 个循环：94 °C 30 秒，52 °C 30 秒，68 °C 30 秒；最后延伸 68 °C 10 分钟。PCR 扩增在 Techne 512 热循环仪上进行。阳性对照为来自**Trichoderma reesei**（DSMZ 编号 768）的 DNA，阴性对照为分子级水。 PCR 后，通过琼脂糖凝胶电泳对扩增子产物和片段大小进行检查。琼脂糖凝胶配方为：1 g 琼脂糖（UltraPure，Invitrogen，美国）、99 mL 1 x TAE（Tris-乙酸-EDTA）缓冲液和 1.6 μL 溴化乙锭（Bio-Rad，美国）。由于 MegaMix Blue 试剂中已含有上样染料，因此无需额外添加。标记物通道加载 2.5 μL HyperLadder 50 bp（BioLine，英国），其包含从 50 bp 到 2.1 kbp 范围内不同大小的 DNA 片段，用于确定 PCR 扩增子片段大小。电泳在 100 V 条件下运行 45 分钟。凝胶通过 UV MultiDoc-It™成像系统（UVP，美国）进行紫外线观察。
Diluted template DNA was then submitted for sequencing on the Illumina MiSeq platform (Illumina, USA) at the NUOMICs Group (Northumbria University, UK). Sequencing was performed targeting the fungal ITS1 region following the EMP Protocol [20]. Sequencing produced 255,617 paired-end reads in a demultiplexed FASTQ file. Due to inadequate quality of the reverse reads, only the forward single-end reads were used in subsequent analysis. Sequencing analysis was performed using DADA2 version 1.12 [22] in R version 3.6.0 [23]. Sequences were trimmed at the 5` by 30 nucleotides (nt) with no truncation at the 3` ends, maximum expected error set to three, and all other options at default. After chimera detection, a total of 19 amplicon sequence variants (ASVs) were identified from 208,584 reads. ASV representative sequences were classified taxonomy using the UNITE 8.0 [24] reference database general FASTA in DADA2. Taxonomic bar plot was generated using PhyloSeq [25].
稀释后的模板 DNA 随后在 NUOMICs 集团（英国诺森比亚大学）使用 Illumina MiSeq 平台（Illumina，美国）进行测序。测序针对真菌的 ITS1 区域，遵循 EMP 协议[20]。测序生成了一个包含 255,617 条去复用的 FASTQ 文件的双端读序文件。由于反向读序的质量不足，在后续分析中仅使用了正向的单端读序。测序分析使用 R 版本 3.6.0 [23]中的 DADA2 版本 1.12 [22]进行。序列在 5`端修剪了 30 个核苷酸（nt），3`端未进行截断，最大预期错误设置为 3，其他选项均为默认值。经过嵌合体检测后，从 208,584 条读序中共识别出 19 个扩增子序列变体（ASVs）。使用 DADA2 中的 UNITE 8.0 [24]参考数据库通用 FASTA 对 ASV 代表序列进行了分类学归类。分类学柱状图使用 PhyloSeq [25]生成。
稀释后的模板 DNA 随后在 NUOMICs 集团（英国诺森比亚大学）使用 Illumina MiSeq 平台（Illumina，美国）进行测序。测序针对真菌的 ITS1 区域，遵循 EMP 协议[20]。测序生成了一个包含 255,617 条去复用的 FASTQ 文件的双端读序文件。由于反向读序的质量不足，在后续分析中仅使用了正向的单端读序。测序分析使用 R 版本 3.6.0 [23]中的 DADA2 版本 1.12 [22]进行。序列在 5`端修剪了 30 个核苷酸（nt），3`端未进行截断，最大预期错误设置为 3，其他选项均为默认值。经过嵌合体检测后，从 208,584 条读序中共识别出 19 个扩增子序列变体（ASVs）。使用 DADA2 中的 UNITE 8.0 [24]参考数据库通用 FASTA 对 ASV 代表序列进行了分类学归类。分类学柱状图使用 PhyloSeq [25]生成。
3. Results and discussion
3. 结果与讨论
Overall, under non-sterile and heterotrophic conditions, microalgae cultures achieved significantly lower levels of nutrient removal than the microalgae/yeast or yeast cultures (Figs. 1, 2 and 4). The COD in wastewater alone (Table 1) produced very low levels of biomass for all cultivations, however, the addition of up to 10 g/L of glucose maximised both biomass yields and nutrient removal. Although glucose was employed to increase the COD loads, a real application of this technology would envisage the use of organic food waste or wastewater with higher COD loads. Simple sugars such as glucose and fructose needed for heterotrophic growth, could be derived from pre-treating organics via saccharification. Mexico City alone generates 12,500 tonnes of municipal solid wastes a day, 50% of which is organic [26]. In addition to municipal waste, a vast amount of industrial organic waste is produced in the country. Agave is grown extensively in Mexico and is used in tequila production, the residue produced from the process is agave bagasse, 352,200 tonnes of which is generated by the industry per year [27]. The refining of sugarcane, another major crop grown throughout Mexico, also generates 15 million tonnes of organic residue annually [27]. Despite the requirement of an additional carbon source, all heterotrophic systems in this study provided substantial improvements in wastewater treatment performance and a fourfold reduction in cultivation time compared to autotrophic microalgae treatment of wastewater of the same source [28]. Heterotrophic microalgae biomass yields, when using 10 g/L of glucose, were lower than yeast biomass yields, under non-sterile conditions (Figs. 1, 2 and 4).
总体而言，在非无菌和异养条件下，微藻培养的养分去除效率显著低于微藻/酵母或单独酵母培养（图 1、2 和 4）。单独使用废水中的 COD（表 1）仅能实现极低的生物质产量，但添加高达 10 g/L 的葡萄糖可最大化生物质产量和养分去除效率。虽然实验中使用葡萄糖提高了 COD 负荷，但该技术的实际应用设想是利用有机食物垃圾或具有更高 COD 负荷的废水。用于异养生长的简单糖类（如葡萄糖和果糖）可以通过对有机物进行糖化预处理获得。仅墨西哥城每天就产生 12,500 吨城市固体垃圾，其中 50%为有机垃圾[26]。除了城市垃圾外，该国还产生大量的工业有机废弃物。龙舌兰在墨西哥广泛种植，并用于生产龙舌兰酒，其生产过程中产生的残渣是龙舌兰渣，每年该行业会产生 352,200 吨龙舌兰渣[27]。此外，整个墨西哥广泛种植的另一种主要作物甘蔗的精炼过程也每年产生 1,500 万吨有机残渣[27]。尽管需要额外的碳源，本研究中的所有异养系统在废水处理性能上均有显著提升，并将培养时间缩短到与同源废水的自养微藻处理相比的四分之一[28]。在非无菌条件下，使用 10 g/L 葡萄糖的异养微藻生物质产量仍低于酵母的生物质产量（图 1、2 和 4）。
总体而言，在非无菌和异养条件下，微藻培养的养分去除效率显著低于微藻/酵母或单独酵母培养（图 1、2 和 4）。单独使用废水中的 COD（表 1）仅能实现极低的生物质产量，但添加高达 10 g/L 的葡萄糖可最大化生物质产量和养分去除效率。虽然实验中使用葡萄糖提高了 COD 负荷，但该技术的实际应用设想是利用有机食物垃圾或具有更高 COD 负荷的废水。用于异养生长的简单糖类（如葡萄糖和果糖）可以通过对有机物进行糖化预处理获得。仅墨西哥城每天就产生 12,500 吨城市固体垃圾，其中 50%为有机垃圾[26]。除了城市垃圾外，该国还产生大量的工业有机废弃物。龙舌兰在墨西哥广泛种植，并用于生产龙舌兰酒，其生产过程中产生的残渣是龙舌兰渣，每年该行业会产生 352,200 吨龙舌兰渣[27]。此外，整个墨西哥广泛种植的另一种主要作物甘蔗的精炼过程也每年产生 1,500 万吨有机残渣[27]。尽管需要额外的碳源，本研究中的所有异养系统在废水处理性能上均有显著提升，并将培养时间缩短到与同源废水的自养微藻处理相比的四分之一[28]。在非无菌条件下，使用 10 g/L 葡萄糖的异养微藻生物质产量仍低于酵母的生物质产量（图 1、2 和 4）。
Fig. 1. Microalgae cultivation in wastewater. (A) Plot of mean experimental nutrient (nitrate, TAN and orthophosphate) concentration ± standard deviation for microalgae cultures supplemented with 10 g/L of glucose. (B) Plot of mean experimental TSS concentration ± standard deviation for microalgae cultures supplemented with 0, 1, 5 or 10 g/L of glucose. Measurement at day six for 1 g/L glucose does not follow the general trend and may have been affected by sampling. Control reactors contained inoculum and wastewater only (0 g/L glucose). TSS concentrations reported, correspond to the microalgae dry weight. Biological experiments were run in triplicate.
图 1. 污水中的微藻培养。
(A) 微藻培养中硝酸盐、总氨氮（TAN）和正磷酸盐的平均实验浓度 ± 标准偏差，葡萄糖添加量为 10 g/L。
(B) 微藻培养中总悬浮固体（TSS）平均实验浓度 ± 标准偏差，葡萄糖添加量分别为 0、1、5 或 10 g/L。1 g/L 葡萄糖在第 6 天的测量结果未遵循一般趋势，可能受到采样的影响。对照反应器仅含有接种物和污水（0 g/L 葡萄糖）。报告的 TSS 浓度对应于微藻的干重。生物实验以三次重复进行。
Fig. 2. Microalgae/yeast co-cultivation in wastewater. Plots of experimental nutrient (nitrate, TAN and orthophosphate) (A), biomass (B) and carbohydrate/COD (C) concentration ± standard deviation for microalgae and yeast culture supplemented with 10 g/L of glucose and air at a rate of 0.5 VVM. Biological experiments were run in triplicate.
图 2. 废水中微藻/酵母的共培养。实验中营养物质（硝酸盐、TAN 和正磷酸盐）（A）、生物质（B）以及碳水化合物/COD（C）的浓度 ± 标准偏差，针对补充了 10 g/L 葡萄糖和以 0.5 VVM 速度通气的微藻和酵母培养物。生物实验以三次重复进行。
3.1. Microalgae cultivation performance in wastewater
3.1. 废水中微藻的培养性能
Cultures supplemented with 10 g/L of glucose attained the highest mean biomass concentration of 0.98 ± 0.10 g/L as shown in Fig. 1B. Resulting p-values from comparing TSS produced in 1, 5 and 10 g/L of glucose to 0 g/L of glucose were 0.46, 0.11 and 0.04, respectively. It can therefore be inferred that only 10 g/L of glucose was sufficient to produce a significant increase in TSS concentration compared to wastewater alone. Wastewater treatment performance was also greatest in cultures supplemented with 10 g/L of glucose achieving mean nitrate, TAN and orthophosphate removals of 60, 53 and 46% respectively as shown in Fig. 1A. However, this is considerably lower than the minimum reductions in total nitrogen and phosphate concentrations of 70 and 80% required to meet the standards outlined in the European Urban Wastewater Treatment Directive [29]. The maximum biomass concentration of 0.98 ± 0.1 g/L was significantly lower than the 3.46 g/L reported in literature for Scenedesmus sp. supplemented with 10 g/L of glucose [4]. This is likely due to the use of non-sterile wastewater in this work, which could have resulted in excess bacterial growth. As bacterial cells are typically smaller than microalgae, several could pass through with the bulk fluid rather than being retained by the 1.6 μm filters used during the TSS analysis.
用添加了 10 g/L 葡萄糖的培养基获得了最高的平均生物量浓度，为 0.98 ± 0.10 g/L，如图 1B 所示。与 0 g/L 葡萄糖相比，在 1、5 和 10 g/L 葡萄糖条件下产生的 TSS 的 p 值分别为 0.46、0.11 和 0.04。因此，可以推断出只有 10 g/L 葡萄糖足以显著增加 TSS 浓度，相较于仅使用废水的情况。废水处理性能在添加 10 g/L 葡萄糖的培养基中也表现最佳，平均硝酸盐、TAN 和正磷酸盐的去除率分别达到 60%、53%和 46%，如图 1A 所示。然而，这显著低于欧洲城市废水处理指令中规定的总氮和磷浓度最低削减率 70%和 80%的标准 [29]。最高生物量浓度 0.98 ± 0.1 g/L 明显低于文献中报道的 Scenedesmus sp.在添加 10 g/L 葡萄糖条件下达到的 3.46 g/L [4]。这可能是由于本研究使用了非无菌废水，导致了过多的细菌生长。由于细菌细胞通常比微藻小，许多细菌可能随主体液体通过，而不是被 TSS 分析过程中使用的 1.6 μm 过滤器截留。
用添加了 10 g/L 葡萄糖的培养基获得了最高的平均生物量浓度，为 0.98 ± 0.10 g/L，如图 1B 所示。与 0 g/L 葡萄糖相比，在 1、5 和 10 g/L 葡萄糖条件下产生的 TSS 的 p 值分别为 0.46、0.11 和 0.04。因此，可以推断出只有 10 g/L 葡萄糖足以显著增加 TSS 浓度，相较于仅使用废水的情况。废水处理性能在添加 10 g/L 葡萄糖的培养基中也表现最佳，平均硝酸盐、TAN 和正磷酸盐的去除率分别达到 60%、53%和 46%，如图 1A 所示。然而，这显著低于欧洲城市废水处理指令中规定的总氮和磷浓度最低削减率 70%和 80%的标准 [29]。最高生物量浓度 0.98 ± 0.1 g/L 明显低于文献中报道的 Scenedesmus sp.在添加 10 g/L 葡萄糖条件下达到的 3.46 g/L [4]。这可能是由于本研究使用了非无菌废水，导致了过多的细菌生长。由于细菌细胞通常比微藻小，许多细菌可能随主体液体通过，而不是被 TSS 分析过程中使用的 1.6 μm 过滤器截留。
3.2. Microalgae and yeast co-cultivation performance in wastewater
3.2. 微藻和酵母在废水中的共培养性能
The microalgae and yeast co-cultures supplemented with 10 or 20 g/L of glucose showed a high level of nutrient removal achieving 91 and 94% orthophosphate, 93 and 97% nitrate and 93 and 95% TAN removal in three days, respectively. After a further four days of culture, TAN was undetectable in all cultures indicating 100% removal. Nitrate concentration decreased slightly, however, orthophosphate concentration increased. No significant differences between the two batches were found in the mean TAN (p = 0.855, two-tailed t-test), nitrate (p = 0.783) and orthophosphate (p = 0.974) concentrations. It can therefore be concluded that doubling the initial glucose concentration resulted in no significant improvement in nutrient removal. The total biomass concentration at the end of the cultivations were 1.85 ± 0.26 to 2.74 ± 0.43 g/L for the 10 and 20 g/L cultures respectively. However, microscopic analysis of the resulting cultures revealed that the higher glucose concentration favoured yeast and bacterial growth and the mean number of microalgae cells present was 18% lower in the 20 g/L cultures. As a result increasing the glucose concentration is unlikely to improve microalgae yields. Hence, a glucose concentration of 10 g/L was selected for subsequent growth experiments. Results for this 10 g/L batch, summarised in Fig. 2, show that between the first and second days, there was an increase in nitrate concentration from 48.5 ± 5.0 to 70.4 ± 9.8 mg/L. This is likely the result of nitrification by the nitrifying bacteria present in the WWTP effluent. Nitrifying bacteria are chemolithoautotrophic organisms meaning they gain their energy through the oxidation of inorganic nitrogen sources using CO2 as a carbon source. There are two main types of nitrifying bacteria used in wastewater treatment. Ammonia oxidising bacteria oxidise ammonia to nitrite whilst nitrite oxidising bacteria are responsible for the oxidation of nitrite to nitrate. The significant increase in nitrate observed suggests that both types of bacteria were likely present in the reactors. Following the increase in nitrate concentration, a 93% decrease was observed in the subsequent 24 h. The nitrogen concentration at 4.8 ± 1.2 mg/L was lower than the 6.1 ± 2.0 mg/L achieved by the microalgae cultures (Fig. 1A) in eight days. It is therefore suspected that the significantly higher nitrogen consumption rate observed in the co-cultures could be associated with yeast growth. This theory is supported by the clear dominance of the yeast after four days of culture (Fig. 3).
在补充了 10 或 20 g/L 葡萄糖的微藻和酵母共培养体系中，三天内营养去除率分别达到了 91%和 94%的正磷酸盐、93%和 97%的硝酸盐以及 93%和 95%的总氨氮（TAN）。在进一步培养四天后，所有培养体系中的 TAN 均检测不到，表明其去除率为 100%。硝酸盐浓度略有下降，但正磷酸盐浓度有所增加。两个批次在平均 TAN（p = 0.855，双尾 t 检验）、硝酸盐（p = 0.783）和正磷酸盐（p = 0.974）浓度上未发现显著差异。因此可以得出结论，初始葡萄糖浓度的加倍并未显著改善营养去除效果。培养结束时，10 g/L 和 20 g/L 体系的总生物量浓度分别为 1.85 ± 0.26 g/L 至 2.74 ± 0.43 g/L。然而，显微镜分析显示，较高的葡萄糖浓度促进了酵母和细菌的生长，而 20 g/L 体系中的微藻细胞数量平均减少了 18%。因此，提高葡萄糖浓度不太可能提高微藻产量。因此，在后续的生长实验中选择了 10 g/L 的葡萄糖浓度。 10 g/L 批次的结果总结于图 2 中，显示在第一天至第二天之间，硝酸盐浓度从 48.5 ± 5.0 mg/L 增加到 70.4 ± 9.8 mg/L。这可能是污水处理厂（WWTP）出水中硝化细菌的硝化作用所致。硝化细菌是化能自养生物，通过氧化无机氮源并利用 CO₂作为碳源获取能量。污水处理常用的硝化细菌主要有两类：氨氧化细菌将氨氧化为亚硝酸盐，而亚硝酸盐氧化细菌负责将亚硝酸盐氧化为硝酸盐。硝酸盐浓度的显著增加表明，这两类细菌可能都存在于反应器中。硝酸盐浓度增加后，在接下来的 24 小时内观察到 93%的下降。氮浓度降至 4.8 ± 1.2 mg/L，低于微藻培养体系在八天内达到的 6.1 ± 2.0 mg/L（图 1A）。因此，推测共培养体系中显著较高的氮消耗率可能与酵母的生长有关。这一推论得到了培养四天后酵母明显占优势的现象（图 3）的支持。
在补充了 10 或 20 g/L 葡萄糖的微藻和酵母共培养体系中，三天内营养去除率分别达到了 91%和 94%的正磷酸盐、93%和 97%的硝酸盐以及 93%和 95%的总氨氮（TAN）。在进一步培养四天后，所有培养体系中的 TAN 均检测不到，表明其去除率为 100%。硝酸盐浓度略有下降，但正磷酸盐浓度有所增加。两个批次在平均 TAN（p = 0.855，双尾 t 检验）、硝酸盐（p = 0.783）和正磷酸盐（p = 0.974）浓度上未发现显著差异。因此可以得出结论，初始葡萄糖浓度的加倍并未显著改善营养去除效果。培养结束时，10 g/L 和 20 g/L 体系的总生物量浓度分别为 1.85 ± 0.26 g/L 至 2.74 ± 0.43 g/L。然而，显微镜分析显示，较高的葡萄糖浓度促进了酵母和细菌的生长，而 20 g/L 体系中的微藻细胞数量平均减少了 18%。因此，提高葡萄糖浓度不太可能提高微藻产量。因此，在后续的生长实验中选择了 10 g/L 的葡萄糖浓度。 10 g/L 批次的结果总结于图 2 中，显示在第一天至第二天之间，硝酸盐浓度从 48.5 ± 5.0 mg/L 增加到 70.4 ± 9.8 mg/L。这可能是污水处理厂（WWTP）出水中硝化细菌的硝化作用所致。硝化细菌是化能自养生物，通过氧化无机氮源并利用 CO₂作为碳源获取能量。污水处理常用的硝化细菌主要有两类：氨氧化细菌将氨氧化为亚硝酸盐，而亚硝酸盐氧化细菌负责将亚硝酸盐氧化为硝酸盐。硝酸盐浓度的显著增加表明，这两类细菌可能都存在于反应器中。硝酸盐浓度增加后，在接下来的 24 小时内观察到 93%的下降。氮浓度降至 4.8 ± 1.2 mg/L，低于微藻培养体系在八天内达到的 6.1 ± 2.0 mg/L（图 1A）。因此，推测共培养体系中显著较高的氮消耗率可能与酵母的生长有关。这一推论得到了培养四天后酵母明显占优势的现象（图 3）的支持。
Fig. 3. Microscope image of microorganisms in wastewater. Microalgae inoculum (A) and microalgae and yeast co-culture supplemented with 10 g/L of glucose on the fourth day of culture (B) Objective values of 100 and 63X, equivalent to total zoom values of 50 and 31.5X were used in A and B respectively. Red plain arrows indicate Scenedesmus sp. whilst the black dotted arrow indicates yeast. Cultures were mixed thoroughly prior to taking samples for microscopic analysis to ensure heterogeneity (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
图 3. 废水中微生物的显微镜图像。微藻接种物（A）以及在培养第四天加入 10 g/L 葡萄糖的微藻与酵母共培养物（B）。A 和 B 分别使用了 100X 和 63X 的物镜，相当于总放大倍数为 50X 和 31.5X。红色实线箭头表示席藻属（Scenedesmus sp.），黑色虚线箭头表示酵母。在进行显微分析取样之前，培养物已充分混合以确保样品的均匀性。（关于图例中颜色引用的说明，请参阅本文的网络版本。）
Fig. 4. Yeast cultivation in wastewater. Plots of experimental nutrient (nitrate, TAN and orthophosphate) (A), biomass (B) and carbohydrate/COD (C) concentration ± standard deviation for yeast batches 1 and 2 cultures supplemented with 10 g/L of glucose and air at a rate of 0.5 VVM. During batch 2 the ethanol concentration was also monitored as shown in (C).
图 4. 酵母在废水中的培养。实验中营养物质（硝酸盐、TAN 和正磷酸盐）（A）、生物质（B）以及碳水化合物/COD（C）的浓度 ± 标准偏差，针对添加了 10 g/L 葡萄糖并以 0.5 VVM 通气的酵母第 1 批和第 2 批培养。在第 2 批培养过程中，还监测了乙醇浓度，如（C）所示。
The total yeast and microalgae biomass concentration decreased from 1.3 ± 0.2 to 1.0 ± 0.1 g/L between days one and two. The poor growth observed may be due to the sub optimal pH conditions. Under sterile conditions Scenedesmus obliquus have been found to grow well at pH 5–9, however, optimal performance is observed between pH 7 and 8 [30]. Yeast are able to tolerate a wide range of pH, however, acidic pH is favourable with growth being slightly reduced at pH 7 and completely arrested at pH 9 [31]. The pH was measured daily and 6 N NaOH was added to raise pH to neutrality. Between measurements and daily pH adjustments, there were considerable drops to around pH 5 in all cultures. The large differences in optimal pH of the two organisms meant that optimal growth of yeast and microalgae in a single reactor may not be feasible if a strict control mechanisms in not in place. Fig. 3A shows the concentrated predominantly microalgae inoculum used to prepare the co-cultures. After just four days of culture the yeast growth became dominant as shown in Fig. 3B. The poor microalgae predominance observed may have been the result of lowered pH as a result of excess yeast growth. The pH dips during growth, are characteristic of fermenting yeasts, such yeasts typically acidify their growth medium through a combination of proton pumping during nutrient uptake, direct organic acid secretion and CO2 evolution and dissolution [32]. Interestingly, the opposite effect was observed when the non-fermenting yeast Lipomyces starkeyi [11] was cultivated alongside microalgae in municipal wastewater. In their case, acidification did not occur and the CO2 product of yeast respiration was utilised in photosynthesis. Although a pH spike promoted autotropic microalgae growth and complete nutrient removal was achieved in 14 days, biomass and hence lipid accumulation were low at 300 and 45 mg/L respectively [11]. The heterotrophic co-cultures in this study achieved comparable wastewater treatment performance and greater biomass accumulation (1.9 ± 0.3 mg/L) in just seven days. TSS concentrations were also significantly higher (p value = 0.004, two-tail t-test) than that of the wastewater controls (0.08 ± 0.02 mg/L).
总酵母和微藻生物量浓度在第一天到第二天之间从 1.3 ± 0.2 g/L 降至 1.0 ± 0.1 g/L。观察到的生长不良可能是由于 pH 条件不佳。在无菌条件下，**Scenedesmus obliquus** 在 pH 5–9 范围内生长良好，但最佳性能出现在 pH 7 和 8 之间[30]。酵母能够耐受广泛的 pH 范围，但酸性 pH 更有利，其生长在 pH 7 时略有减少，在 pH 9 时完全停止[31]。每天测量 pH，并添加 6 N NaOH 将 pH 提高至中性。在测量和每日 pH 调整之间，所有培养物的 pH 都明显下降至约 pH 5。两种生物的最佳 pH 差异较大，这意味着如果没有严格的控制机制，在单一反应器中同时实现酵母和微藻的最佳生长可能不可行。图 3A 显示了用于制备共培养物的以微藻为主的浓缩接种物。仅培养四天后，酵母生长已占主导地位，如图 3B 所示。观察到的微藻未能占主导地位的情况可能是由于酵母过度生长导致的 pH 降低所致。生长期间的 pH 下降是发酵酵母的特征，这类酵母通常通过营养物质摄取过程中的质子泵送、直接有机酸分泌以及 CO₂ 的释放和溶解来使其培养基酸化[32]。有趣的是，当非发酵酵母**Lipomyces starkeyi**[11]与微藻在市政废水中共同培养时，观察到了相反的效果。在他们的研究中，没有发生酸化现象，且酵母呼吸产生的 CO₂ 被用于光合作用。尽管 pH 升高促进了自养微藻的生长，并在 14 天内实现了完全的营养物质去除，但生物量和脂质的积累较低，分别为 300 mg/L 和 45 mg/L[11]。本研究中的异养共培养在仅 7 天内实现了可比的废水处理性能，并获得了更高的生物量积累（1.9 ± 0.3 mg/L）。总悬浮固体（TSS）浓度也显著高于废水对照组（0.08 ± 0.02 mg/L）（双尾 t 检验，p 值 = 0.004）。
总酵母和微藻生物量浓度在第一天到第二天之间从 1.3 ± 0.2 g/L 降至 1.0 ± 0.1 g/L。观察到的生长不良可能是由于 pH 条件不佳。在无菌条件下，**Scenedesmus obliquus** 在 pH 5–9 范围内生长良好，但最佳性能出现在 pH 7 和 8 之间[30]。酵母能够耐受广泛的 pH 范围，但酸性 pH 更有利，其生长在 pH 7 时略有减少，在 pH 9 时完全停止[31]。每天测量 pH，并添加 6 N NaOH 将 pH 提高至中性。在测量和每日 pH 调整之间，所有培养物的 pH 都明显下降至约 pH 5。两种生物的最佳 pH 差异较大，这意味着如果没有严格的控制机制，在单一反应器中同时实现酵母和微藻的最佳生长可能不可行。图 3A 显示了用于制备共培养物的以微藻为主的浓缩接种物。仅培养四天后，酵母生长已占主导地位，如图 3B 所示。观察到的微藻未能占主导地位的情况可能是由于酵母过度生长导致的 pH 降低所致。生长期间的 pH 下降是发酵酵母的特征，这类酵母通常通过营养物质摄取过程中的质子泵送、直接有机酸分泌以及 CO₂ 的释放和溶解来使其培养基酸化[32]。有趣的是，当非发酵酵母**Lipomyces starkeyi**[11]与微藻在市政废水中共同培养时，观察到了相反的效果。在他们的研究中，没有发生酸化现象，且酵母呼吸产生的 CO₂ 被用于光合作用。尽管 pH 升高促进了自养微藻的生长，并在 14 天内实现了完全的营养物质去除，但生物量和脂质的积累较低，分别为 300 mg/L 和 45 mg/L[11]。本研究中的异养共培养在仅 7 天内实现了可比的废水处理性能，并获得了更高的生物量积累（1.9 ± 0.3 mg/L）。总悬浮固体（TSS）浓度也显著高于废水对照组（0.08 ± 0.02 mg/L）（双尾 t 检验，p 值 = 0.004）。
3.3. Yeast cultivation performance in wastewater
3.3. 酵母在废水中的培养性能
Yeast were found to thrive in the municipal wastewater successfully outcompeting bacteria under non-sterile heterotrophic conditions and produce consistently high nutrient removal in line with European standards. Results of the first two batches of triplicate yeast cultures are summarised in Fig. 4. During the first three days of batch 1, 94% TAN, 90% orthophosphate, 40% nitrate, 95% carbohydrate and 50% COD removal was achieved. In the subsequent five days of culture, the nitrate and TAN concentrations decreased at a slower rate until undetectable suggesting 100% removal (Fig. 4). Nitrate assimilation, a relatively uncommon characteristic in yeast, was observed in all yeast cultures. The inability of industrial yeast strains to assimilate nitrate could be the reason municipal wastewater treatment using yeast alone is yet to have been explored in detail. A slight increase in orthophosphate concentration was seen before a decrease resulting in a final removal of 93%. A final removal of 97% carbohydrates and COD was also observed following eight days of culture.
酵母在市政废水中能够成功生长，在非无菌异养条件下优于细菌，并实现了符合欧洲标准的一致性高效养分去除。图 4 总结了前两组三重复酵母培养的结果。在第一批培养的前三天内，氨氮（TAN）去除率为 94%，正磷酸盐去除率为 90%，硝酸盐去除率为 40%，碳水化合物去除率为 95%，化学需氧量（COD）去除率为 50%。在随后的五天培养中，硝酸盐和氨氮浓度以较慢速度下降，直到不可检测，表明实现了 100%的去除（见图 4）。在所有酵母培养中观察到了硝酸盐同化现象，这在酵母中是相对少见的特性。工业酵母菌株无法同化硝酸盐，可能是用酵母单独处理市政废水尚未被详细探索的原因。在正磷酸盐浓度下降之前，曾观察到其略有增加，最终去除率达到 93%。经过八天的培养，碳水化合物和 COD 的最终去除率分别达到了 97%。
酵母在市政废水中能够成功生长，在非无菌异养条件下优于细菌，并实现了符合欧洲标准的一致性高效养分去除。图 4 总结了前两组三重复酵母培养的结果。在第一批培养的前三天内，氨氮（TAN）去除率为 94%，正磷酸盐去除率为 90%，硝酸盐去除率为 40%，碳水化合物去除率为 95%，化学需氧量（COD）去除率为 50%。在随后的五天培养中，硝酸盐和氨氮浓度以较慢速度下降，直到不可检测，表明实现了 100%的去除（见图 4）。在所有酵母培养中观察到了硝酸盐同化现象，这在酵母中是相对少见的特性。工业酵母菌株无法同化硝酸盐，可能是用酵母单独处理市政废水尚未被详细探索的原因。在正磷酸盐浓度下降之前，曾观察到其略有增加，最终去除率达到 93%。经过八天的培养，碳水化合物和 COD 的最终去除率分别达到了 97%。
The metabolism of yeast is highly dependent on aeration, the nature and concentration of substrate and on the type of yeast present. Many yeast strains such as Kluyveromyces marxianus are Crabtree negative meaning that under aerobic conditions [33], glucose metabolism occurs via aerobic respiration only and fermentation does not occur. Some yeast strains such as Saccharomyces cerevisiae, however, are Crabtree positive [34]. This type of yeast typically undergoes two sequential exponential growth phases in glucose-limited cultures [34]. Crabtree positive growth was observed in this study as shown in Fig. 4. An exponential growth phase was observed during the first two days of culture where biomass concentration increased from 0.2 to 2.1 ± 0.1 g/L as shown in Fig. 4B. This was accompanied by a rapid depletion in nutrients (Fig. 4A). During this period glucose is in abundance and the yeast cells derive their energy from fermentation through the glycolytic pathway producing ethanol and CO2. The COD is significantly higher than the carbohydrate concentration on day two at 7363 ± 279 compared to 812 ± 103 as shown in Fig. 4C. This indicates the presence of another organic carbon source (e.g. ethanol) not present at the beginning of the culture. Once the glucose supply has been exhausted, the cells enter a diauxic shift characterised by a short stationary phase during which the metabolism is switched to aerobic respiration. This can be seen for batch 1 between days two and three in Fig. 4B by the slight increase in concentration to 2.4 ± 0.1 g/L. The concentration of carbohydrates decreased from 10,012 ± 0.2 to 490 ± 39 mg/L in the first three days of culture as shown in Fig. 4C. A second exponential growth phase occurs after this point once more than 95% of the glucose in the culture medium had been consumed. This can be seen by the rise in biomass concentration to 3.3 ± 0.1 g/L on day four in Fig. 4B. Finally, a second stationary phase is reached once the COD had sufficiently depleted to 0.3 ± 0.3 g/L. In order to validate the theory that the yeast present were in fact Crabtree positive an additional batch of cultures (batch 2) was ran under the same conditions and ethanol concentration was monitored (Fig. 4). Despite batch to batch variations in initial nutrient concentrations due to the nature of the culture medium, a high level of repeatability was observed as shown in Fig. 4A. During batch 2, a peak ethanol concentration of 1750 mg/L occurred on the second day as shown Fig. 4C, only a slight increase from the previous day’s value of 1690 mg/L despite a decrease in carbohydrate concentration of 7145 mg/L. In addition, the stationary phase of growth cannot be clearly seen in Fig. 4B. Carbohydrate concentration had greatly depleted by day two and exponential growth was seen between days two and four, suggesting that the diauxic shift and hence peak ethanol concentration may have occurred between the measurements taken on the first and second days.
酵母的代谢高度依赖于供氧、底物的性质和浓度以及酵母菌株的类型。许多酵母菌株（如 Kluyveromyces marxianus）是 Crabtree 阴性，这意味着在有氧条件下[33]，葡萄糖仅通过有氧呼吸代谢，不发生发酵。然而，某些酵母菌株（如 Saccharomyces cerevisiae）是 Crabtree 阳性[34]。这种类型的酵母在葡萄糖有限的培养中通常经历两个连续的指数生长期[34]。在本研究中观察到了 Crabtree 阳性生长，如图 4 所示。在培养的前两天，观察到了指数生长期，生物量浓度从 0.2 g/L 增加到 2.1 ± 0.1 g/L（图 4B），同时伴随着养分的快速耗尽（图 4A）。在此期间，葡萄糖充足，酵母细胞通过糖酵解途径进行发酵以产生乙醇和 CO 2 来获取能量。在第二天，化学需氧量（COD）显著高于碳水化合物浓度，分别为 7363 ± 279 和 812 ± 103（图 4C），这表明存在另一种在培养初期不存在的有机碳源（如乙醇）。 一旦葡萄糖耗尽，细胞进入二相生长的转换期（diauxic shift），其特征是一个短暂的停滞期，此期间代谢切换为有氧呼吸。对此，可通过图 4B 中第 1 组培养在第二天至第三天的生物量浓度轻微增加至 2.4 ± 0.1 g/L 观察到。培养的前三天，碳水化合物浓度从 10,012 ± 0.2 mg/L 下降到 490 ± 39 mg/L（图 4C）。在此之后，当培养基中超过 95%的葡萄糖被消耗时，发生第二次指数生长期。此过程可通过第四天生物量浓度上升至 3.3 ± 0.1 g/L 观察到（图 4B）。最后，当化学需氧量（COD）显著下降至 0.3 ± 0.3 g/L 时，培养进入第二个稳定期。 为了验证酵母确实是 Crabtree 阳性，在相同条件下进行了另一组培养（第 2 组），并监测了乙醇浓度（图 4）。尽管由于培养基性质导致初始养分浓度存在批次间差异，但如图 4A 所示，仍观察到了较高的重复性。在第 2 组培养中，第二天的乙醇浓度峰值为 1750 mg/L，与前一天的 1690 mg/L 相比仅有小幅增加，尽管碳水化合物浓度减少了 7145 mg/L。此外，在图 4B 中未能清晰观察到生长停滞期。第二天碳水化合物浓度已经大幅下降，并且在第二天至第四天间观察到指数生长，这表明二相生长转换期以及乙醇浓度峰值可能发生在第一天和第二天的测量时间点之间。
酵母的代谢高度依赖于供氧、底物的性质和浓度以及酵母菌株的类型。许多酵母菌株（如 Kluyveromyces marxianus）是 Crabtree 阴性，这意味着在有氧条件下[33]，葡萄糖仅通过有氧呼吸代谢，不发生发酵。然而，某些酵母菌株（如 Saccharomyces cerevisiae）是 Crabtree 阳性[34]。这种类型的酵母在葡萄糖有限的培养中通常经历两个连续的指数生长期[34]。在本研究中观察到了 Crabtree 阳性生长，如图 4 所示。在培养的前两天，观察到了指数生长期，生物量浓度从 0.2 g/L 增加到 2.1 ± 0.1 g/L（图 4B），同时伴随着养分的快速耗尽（图 4A）。在此期间，葡萄糖充足，酵母细胞通过糖酵解途径进行发酵以产生乙醇和 CO 2 来获取能量。在第二天，化学需氧量（COD）显著高于碳水化合物浓度，分别为 7363 ± 279 和 812 ± 103（图 4C），这表明存在另一种在培养初期不存在的有机碳源（如乙醇）。 一旦葡萄糖耗尽，细胞进入二相生长的转换期（diauxic shift），其特征是一个短暂的停滞期，此期间代谢切换为有氧呼吸。对此，可通过图 4B 中第 1 组培养在第二天至第三天的生物量浓度轻微增加至 2.4 ± 0.1 g/L 观察到。培养的前三天，碳水化合物浓度从 10,012 ± 0.2 mg/L 下降到 490 ± 39 mg/L（图 4C）。在此之后，当培养基中超过 95%的葡萄糖被消耗时，发生第二次指数生长期。此过程可通过第四天生物量浓度上升至 3.3 ± 0.1 g/L 观察到（图 4B）。最后，当化学需氧量（COD）显著下降至 0.3 ± 0.3 g/L 时，培养进入第二个稳定期。 为了验证酵母确实是 Crabtree 阳性，在相同条件下进行了另一组培养（第 2 组），并监测了乙醇浓度（图 4）。尽管由于培养基性质导致初始养分浓度存在批次间差异，但如图 4A 所示，仍观察到了较高的重复性。在第 2 组培养中，第二天的乙醇浓度峰值为 1750 mg/L，与前一天的 1690 mg/L 相比仅有小幅增加，尽管碳水化合物浓度减少了 7145 mg/L。此外，在图 4B 中未能清晰观察到生长停滞期。第二天碳水化合物浓度已经大幅下降，并且在第二天至第四天间观察到指数生长，这表明二相生长转换期以及乙醇浓度峰值可能发生在第一天和第二天的测量时间点之间。
Further growth experiments were conducted under the same conditions allowing ethanol concentration to be monitored more accurately as shown in Fig. 5. There was a short lag phase within the first 2.5 h in which ethanol concentration rose slightly from 0 to 0.3 g/L. Following this, the ethanol concentration increased exponentially to 1.6 g/L by hour six. There was then a much slower increase to the peak value of 2.5 g/L in the subsequent 42 h.
在相同条件下进行了进一步的生长实验，以更准确地监测乙醇浓度，如图 5 所示。在最初的 2.5 小时内存在一个短暂的迟滞期，此期间乙醇浓度从 0 稍微升高至 0.3 g/L。随后，乙醇浓度呈指数增长，在第 6 小时达到 1.6 g/L。之后，浓度增长显著放缓，在接下来的 42 小时内缓慢增加至峰值 2.5 g/L。
在相同条件下进行了进一步的生长实验，以更准确地监测乙醇浓度，如图 5 所示。在最初的 2.5 小时内存在一个短暂的迟滞期，此期间乙醇浓度从 0 稍微升高至 0.3 g/L。随后，乙醇浓度呈指数增长，在第 6 小时达到 1.6 g/L。之后，浓度增长显著放缓，在接下来的 42 小时内缓慢增加至峰值 2.5 g/L。
Fig. 5. Yeast cultivation in wastewater to monitor bioethanol production. Plot of experimental ethanol, biomass and carbohydrate concentration for yeast batch 3 cultures supplemented with 10 g/L of glucose.
图 5. 在废水中培养酵母以监测生物乙醇的生产。酵母第 3 批培养物中添加 10 g/L 葡萄糖的实验乙醇、菌体和碳水化合物浓度曲线图。
At the end of the 48 h the carbohydrate concentration had depleted from 10,006 ± 48 to just 266 ± 74 mg/L. In addition, between 30 and 48 h the biomass concentration increased only slightly from 2.0 ± 0.1 to 2.3 ± 0.6 g/L suggesting that the cells were in the diauxic phase of growth as expected. The peak ethanol concentration was 2490 mg/L, corresponding to an ethanol yield of 25% by weight. This is lower than the literature yield of 32% for yeast grown aerobically in a sterile medium [34]. One possible cause of the lower than expected yield is the lack of pH control. Given the initial pH of the wastewater was around neutral which is optimal for bacteria whereas acidic pH favours yeast growth. By controlling pH in the early stages of microbial growth, bacterial growth could have been supressed and the availability of glucose for ethanol production increased. In addition, increasing the dissolved oxygen concentration beyond 13% in Saccharomyces cerevisiae cultures has been found to reduce ethanol yields [35]. Excess aeration of the cultures may have also hindered ethanol production.
在 48 小时结束时，碳水化合物浓度从 10,006 ± 48 mg/L 减少到仅 266 ± 74 mg/L。此外，在 30 至 48 小时之间，生物质浓度仅从 2.0 ± 0.1 g/L 略微增加到 2.3 ± 0.6 g/L，这表明细胞处于预期的双相生长阶段。最大乙醇浓度为 2490 mg/L，对应的乙醇产率为 25%（按重量计）。这低于文献中在无菌培养基中需氧生长酵母的产率 32% [34]。导致产率低于预期的一个可能原因是缺乏 pH 控制。鉴于废水的初始 pH 值接近中性，这对于细菌是最优的，而酸性 pH 更有利于酵母的生长。在微生物生长的早期阶段控制 pH 值可以抑制细菌的生长，从而增加用于乙醇生产的葡萄糖的可用性。此外，在酿酒酵母（Saccharomyces cerevisiae）培养物中将溶解氧浓度提高到 13%以上被发现会降低乙醇产率[35]。培养物的过度通气也可能阻碍了乙醇的生产。
在 48 小时结束时，碳水化合物浓度从 10,006 ± 48 mg/L 减少到仅 266 ± 74 mg/L。此外，在 30 至 48 小时之间，生物质浓度仅从 2.0 ± 0.1 g/L 略微增加到 2.3 ± 0.6 g/L，这表明细胞处于预期的双相生长阶段。最大乙醇浓度为 2490 mg/L，对应的乙醇产率为 25%（按重量计）。这低于文献中在无菌培养基中需氧生长酵母的产率 32% [34]。导致产率低于预期的一个可能原因是缺乏 pH 控制。鉴于废水的初始 pH 值接近中性，这对于细菌是最优的，而酸性 pH 更有利于酵母的生长。在微生物生长的早期阶段控制 pH 值可以抑制细菌的生长，从而增加用于乙醇生产的葡萄糖的可用性。此外，在酿酒酵母（Saccharomyces cerevisiae）培养物中将溶解氧浓度提高到 13%以上被发现会降低乙醇产率[35]。培养物的过度通气也可能阻碍了乙醇的生产。
Phosphorous in the form of inorganic phosphate is one of the most important metabolites for all organisms [36]; it is involved in the biosynthesis of many cellular components such as ATP, nucleic acids, phospholipids and proteins. In the first two yeast batches the mean orthophosphate concentration depleted rapidly to below 10 mg/L, following this there was an increase to 11.8 ± 1.1 and 10.6 ± 1.4 mg/L in the first and second batches, respectively. The orthophosphate concentration then decreased to final values of 5.6 ± 0.9 and 8.7 ± 0.7 mg/L at the end of the eight and seven day batches, respectively. Orthophosphate concentration corresponds to the concentration of free phosphate (PO43−) ions in the culture medium rather than the total phosphate content. Studies on yeast cells have shown that regulation of the expression of genes involved in phosphate metabolism is dependent on the phosphate concentration of the external medium [36]. The depletion of phosphate from the culture medium can result in up to a 130-fold increase in ectophosphatase activity [36]. Ectophospatases hydrolyse organic phosphates releasing additional phosphate molecules. This could have caused the slight increases in orthophosphate concentration observed in all yeast experiments.
无机磷酸盐形式的磷是所有生物体中最重要的代谢物之一 [36]；它参与许多细胞成分的生物合成，如 ATP、核酸、磷脂和蛋白质。在前两批酵母实验中，平均正磷酸盐浓度迅速下降到低于 10 mg/L，随后在第一批和第二批中分别增加到 11.8 ± 1.1 mg/L 和 10.6 ± 1.4 mg/L。之后，正磷酸盐浓度在第八天和第七天的实验结束时分别降至 5.6 ± 0.9 mg/L 和 8.7 ± 0.7 mg/L 的最终值。正磷酸盐浓度对应于培养基中自由磷酸盐（PO₄³⁻）离子的浓度，而不是总磷含量。针对酵母细胞的研究表明，磷酸盐代谢相关基因的表达调控依赖于外部培养基中的磷酸盐浓度 [36]。培养基中磷酸盐的消耗可能导致外磷酸酶活性增加多达 130 倍 [36]。外磷酸酶通过水解有机磷酸盐释放出额外的磷酸盐分子，这可能是所有酵母实验中观察到正磷酸盐浓度轻微增加的原因。
无机磷酸盐形式的磷是所有生物体中最重要的代谢物之一 [36]；它参与许多细胞成分的生物合成，如 ATP、核酸、磷脂和蛋白质。在前两批酵母实验中，平均正磷酸盐浓度迅速下降到低于 10 mg/L，随后在第一批和第二批中分别增加到 11.8 ± 1.1 mg/L 和 10.6 ± 1.4 mg/L。之后，正磷酸盐浓度在第八天和第七天的实验结束时分别降至 5.6 ± 0.9 mg/L 和 8.7 ± 0.7 mg/L 的最终值。正磷酸盐浓度对应于培养基中自由磷酸盐（PO₄³⁻）离子的浓度，而不是总磷含量。针对酵母细胞的研究表明，磷酸盐代谢相关基因的表达调控依赖于外部培养基中的磷酸盐浓度 [36]。培养基中磷酸盐的消耗可能导致外磷酸酶活性增加多达 130 倍 [36]。外磷酸酶通过水解有机磷酸盐释放出额外的磷酸盐分子，这可能是所有酵母实验中观察到正磷酸盐浓度轻微增加的原因。
TAN removal by the yeast was very efficient with over 90% removal in the first three days and 100% removal by the end of the two batches. Nitrification was much less prominent in the yeast cultures than the equivalent co-cultures. This may have been due to a low pH. The two commonly used nitrifying bacteria, Nitrosomonas and Nitrobacter perform nitrification over a pH range of 6.5–10 [37]. The pH of the yeast cultures dropped to around 4.5, significantly lower than the minimum tolerable pH of 6.5 which is likely to have inhibited nitrification. Also, as most bacteria thrive at neutral pH, the low pH as a result of acidification by the yeast cells is likely to have supressed bacterial growth in the yeast cultures. This is desirable as excess bacterial growth may hinder nutrient removal and recovery of the yeast cells following treatment. The yeast presented good flocculation properties following treatment of the wastewater. Following cultivation contents of the bioreactors were stored at 4 °C for three days prior to yeast extraction. Within 24 h of storage time, bulk sedimentation occurred facilitating 74% separation of the yeast cells without centrifugation.
酵母对 TAN 的去除效率非常高，在前三天内去除率超过 90%，到两个批次结束时去除率达到 100%。在酵母培养物中，硝化作用远不如相应的共培养物明显，这可能是由于较低的 pH 值所致。两种常见的硝化细菌 Nitrosomonas 和 Nitrobacter 在 pH 范围为 6.5-10 时进行硝化[37]。而酵母培养物的 pH 值降至约 4.5，显著低于其最低可耐受的 pH 值 6.5，这可能抑制了硝化作用。此外，由于大多数细菌在中性 pH 条件下繁殖良好，而酵母细胞引起的酸化导致的低 pH 值可能抑制了酵母培养物中的细菌生长。这是理想的，因为过度的细菌生长可能会妨碍营养物质的去除以及处理后酵母细胞的回收。酵母在废水处理后表现出了良好的絮凝特性。在培养后，生物反应器的内容物在 4°C 下储存三天，然后提取酵母细胞。在储存后的 24 小时内发生了大量沉降，无需离心即可实现 74%的酵母细胞分离。
酵母对 TAN 的去除效率非常高，在前三天内去除率超过 90%，到两个批次结束时去除率达到 100%。在酵母培养物中，硝化作用远不如相应的共培养物明显，这可能是由于较低的 pH 值所致。两种常见的硝化细菌 Nitrosomonas 和 Nitrobacter 在 pH 范围为 6.5-10 时进行硝化[37]。而酵母培养物的 pH 值降至约 4.5，显著低于其最低可耐受的 pH 值 6.5，这可能抑制了硝化作用。此外，由于大多数细菌在中性 pH 条件下繁殖良好，而酵母细胞引起的酸化导致的低 pH 值可能抑制了酵母培养物中的细菌生长。这是理想的，因为过度的细菌生长可能会妨碍营养物质的去除以及处理后酵母细胞的回收。酵母在废水处理后表现出了良好的絮凝特性。在培养后，生物反应器的内容物在 4°C 下储存三天，然后提取酵母细胞。在储存后的 24 小时内发生了大量沉降，无需离心即可实现 74%的酵母细胞分离。
As yeast was found to be present in the municipal wastewater used, reactors containing just wastewater supplemented with 10 g/L of glucose were monitored to determine the effect of initial inoculation. The control culture achieved nitrate, TAN and orthophosphate removals of 73, 78 and 86% respectively in seven days. However, in this case the yeast struggled to compete with bacteria. A two-tailed two-sample t-test detected a significant difference between the means of the biomass concentrations observed for the inoculated and control cultures (p = 0.05). However, there were no significant differences observed in the TAN (p = 0.572), nitrate (p = 0.374) and orthophosphate (p = 0.423) removals. Whilst inoculating the cultures did not result in a significant increase in nutrient removal, it did prevent excessive bacterial growth. The large amount of bacteria present in the control cultures was undesirable as it resulted in a lower yield of yeast and may hinder cell recovery. As the yeast cells could generate a higher revenue than low-value bacterial biomass this would also likely reduce the profitability of the process. The addition of a small amount of yeast to the culture medium (0.2 g/L) resulted in a 175% increase in final yeast biomass concentration.
由于在使用的市政废水中发现了酵母，研究人员监测了仅添加了 10 g/L 葡萄糖的废水反应器，以确定初始接种的影响。对照组在 7 天内分别实现了 73%、78%和 86%的硝酸盐、总氨氮（TAN）和正磷酸盐去除率。然而，在这种情况下，酵母在与细菌的竞争中表现不佳。双尾两样本 t 检验检测到接种组和对照组生物量浓度均值之间存在显著差异（p = 0.05）。然而，在总氨氮（p = 0.572）、硝酸盐（p = 0.374）和正磷酸盐（p = 0.423）去除率上未观察到显著差异。虽然接种培养物未显著提高营养物去除率，但有效抑制了细菌的过度生长。对照组中大量存在的细菌是不理想的，因为它导致酵母产量降低，并可能阻碍细胞回收。由于酵母细胞的经济价值高于低价值的细菌生物量，这也可能降低工艺的盈利能力。在培养基中加入少量酵母（0.2 g/L）使最终酵母生物量浓度增加了 175%。
由于在使用的市政废水中发现了酵母，研究人员监测了仅添加了 10 g/L 葡萄糖的废水反应器，以确定初始接种的影响。对照组在 7 天内分别实现了 73%、78%和 86%的硝酸盐、总氨氮（TAN）和正磷酸盐去除率。然而，在这种情况下，酵母在与细菌的竞争中表现不佳。双尾两样本 t 检验检测到接种组和对照组生物量浓度均值之间存在显著差异（p = 0.05）。然而，在总氨氮（p = 0.572）、硝酸盐（p = 0.374）和正磷酸盐（p = 0.423）去除率上未观察到显著差异。虽然接种培养物未显著提高营养物去除率，但有效抑制了细菌的过度生长。对照组中大量存在的细菌是不理想的，因为它导致酵母产量降低，并可能阻碍细胞回收。由于酵母细胞的经济价值高于低价值的细菌生物量，这也可能降低工艺的盈利能力。在培养基中加入少量酵母（0.2 g/L）使最终酵母生物量浓度增加了 175%。
After 48 h of growth, the mean biomass concentrations of each of the three yeast batches were 2.1 ± 0.1, 2.8 ± 0.2 and 2.3 ± 0.6 g/L, respectively (Figs. 4,5). A test for equal variances on the final TSS concentrations of the three batches showed that there was no significant difference (p = 0.881), meaning high reproducibility of yeast yields obtained when using different batches of non-sterilised wastewater.
在生长 48 小时后，三组酵母的平均生物量浓度分别为 2.1 ± 0.1、2.8 ± 0.2 和 2.3 ± 0.6 g/L（图 4、图 5）。对三组最终 TSS 浓度进行等方差检验显示无显著差异（p = 0.881），这表明在使用不同批次未灭菌废水时，酵母产量具有较高的可重复性。
在生长 48 小时后，三组酵母的平均生物量浓度分别为 2.1 ± 0.1、2.8 ± 0.2 和 2.3 ± 0.6 g/L（图 4、图 5）。对三组最终 TSS 浓度进行等方差检验显示无显著差异（p = 0.881），这表明在使用不同批次未灭菌废水时，酵母产量具有较高的可重复性。
3.4. Yeast identification and use for wastewater treatment
3.4. 酵母的鉴定及其在废水处理中的应用
The most abundant fungal taxonomic group identified in ITS sequencing was the Saccharomycetes species Barnettomyza californica, which accounted for 45.4% of the total abundance of the sample (Fig. 6). The Saccharomycetes species Cyberlindnera subsufficiens accounted for 29.3%, whilst a Candida sp. accounted for 23.5%. Since the Candida sequence did not classify to species level, a BLAST search of the representative sequence was performed, which resulted in significant alignments with Scheffersomyces spartinae, Candida thasaenensis, Candida gosingica, all with a score of 388 at 80% query coverage and 98.6% identity. No other representative sequence accounted for more than 0.1% of abundance. Barnettomyza californica and Cyberlindnera subsufficiens, the two species which accounted for around 75% of abundance are both known to be capable of both glucose fermentation and nitrate assimilation [38].
在 ITS 测序中鉴定出的最丰富的真菌分类群是 Saccharomycetes 纲的加州巴奈酵母（*Barnettomyza californica*），占样本总丰度的 45.4%（图 6）。Saccharomycetes 纲的次充酵母（*Cyberlindnera subsufficiens*）占 29.3%，而一个未分类的念珠菌属（*Candida* sp.）占 23.5%。由于该念珠菌序列未被分类到种级，因此对其代表序列进行了 BLAST 搜索，结果显示与以下物种有显著比对结果：斯巴达酵母（*Scheffersomyces spartinae*）、塔塞念珠菌（*Candida thasaenensis*）、高兴念珠菌（*Candida gosingica*），其得分均为 388，查询覆盖率为 80%，相似性为 98.6%。没有其他代表序列的丰度超过 0.1%。加州巴奈酵母和次充酵母这两个占总丰度约 75%的物种已知均具有葡萄糖发酵和硝酸盐同化的能力[38]。
在 ITS 测序中鉴定出的最丰富的真菌分类群是 Saccharomycetes 纲的加州巴奈酵母（*Barnettomyza californica*），占样本总丰度的 45.4%（图 6）。Saccharomycetes 纲的次充酵母（*Cyberlindnera subsufficiens*）占 29.3%，而一个未分类的念珠菌属（*Candida* sp.）占 23.5%。由于该念珠菌序列未被分类到种级，因此对其代表序列进行了 BLAST 搜索，结果显示与以下物种有显著比对结果：斯巴达酵母（*Scheffersomyces spartinae*）、塔塞念珠菌（*Candida thasaenensis*）、高兴念珠菌（*Candida gosingica*），其得分均为 388，查询覆盖率为 80%，相似性为 98.6%。没有其他代表序列的丰度超过 0.1%。加州巴奈酵母和次充酵母这两个占总丰度约 75%的物种已知均具有葡萄糖发酵和硝酸盐同化的能力[38]。
Fig. 6. Yeast Identification. Stacked bar chart of fractional abundance of the three representative sequences that accounted for 98.2% of the total sequenced community, as derived from DADA2. The size of each section of the bar reflects the relative fractional abundance of a given representative sequence as a proportion of the total read count for the three sequences. Red denotes Barnettozyma californica (45.4%); blue denotes Cyberlindnera subsufficiens (29.3%); beige denotes Candida sp. (23.5%). No other representative sequences accounted for > 0.1% abundance and so were excluded from this figure (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
图 6. 酵母鉴定。堆叠柱状图显示了三个代表性序列的分数丰度，这三个序列占总测序群落的 98.2%，数据来源于 DADA2 分析。柱状图中每个部分的大小表示某个代表性序列在三个序列总读取数中的相对分数丰度。红色表示加州巴奈托酵母（45.4%）；蓝色表示次充盈赛百霉酵母（29.3%）；米色表示念珠菌属（23.5%）。没有其他代表性序列的丰度超过 0.1%，因此未列入此图。（关于图例中颜色的解释，请参阅本文网络版）。
Wild wastewater yeast cultivations present a relatively low cost means of treating municipal wastewater. With careful selection of the microorganisms present there is potential to greatly improve yields of the desired biofuel. The use of a non-fermenting nitrate assimilating yeast such as Lipomyces starkeyi could improve nutrient removal without the acidification effect associated with fermenting yeasts. This would allow alkaline pH conditions to be maintained promoting microalgal growth and lipid accumulation. On the other hand, increasing the proportion of brewing yeasts in the fermentation medium would maximise bioethanol yields.
野生废水酵母培养是一种相对低成本的市政废水处理方法。通过仔细选择其中的微生物，有可能显著提高目标生物燃料的产量。使用像 Lipomyces starkeyi 这样的非发酵硝酸盐同化酵母，可以在不引起发酵酵母相关酸化效应的情况下改善养分去除。这将使碱性 pH 条件得以维持，从而促进微藻的生长和脂质的积累。另一方面，增加发酵培养基中酿酒酵母的比例可以最大化生物乙醇的产量。
野生废水酵母培养是一种相对低成本的市政废水处理方法。通过仔细选择其中的微生物，有可能显著提高目标生物燃料的产量。使用像 Lipomyces starkeyi 这样的非发酵硝酸盐同化酵母，可以在不引起发酵酵母相关酸化效应的情况下改善养分去除。这将使碱性 pH 条件得以维持，从而促进微藻的生长和脂质的积累。另一方面，增加发酵培养基中酿酒酵母的比例可以最大化生物乙醇的产量。
4. Conclusions 4. 结论
An investigation into the performance of novel non-sterile heterotrophic systems to remove nutrients and organic matter from municipal wastewater has been conducted. Glucose supplementation was necessary in this study to deplete nutrients and increase biomass yields, hence the system proposed could be of interest to the industrial sector providing a sustainable means of treating wastewaters with high organic loads or pre-treated organic waste. Microalgae were unable to compete with proliferating microorganisms, achieving relatively low biomass concentrations along with poor levels of nutrient removal. Co-culturing microalgae with yeast led to poor yields of both microalgae and yeast mainly due to pH fluctuations, however, excellent levels of nutrient removal were achieved. The wild yeast consortium found in the municipal wastewater produced the most promising results, achieving consistently high biomass concentrations and nutrient removal rates. Here, nitrate assimilation, a relatively uncommon characteristic in yeast, maximised total nitrogen removal in the yeast cultures; and the Crabtree positive metabolism of the wild yeast consortium highlighted the possibility for simultaneous wastewater treatment and bioethanol production. With this, our study showed the potential for generating useful yeast biomass and bioethanol whilst achieving wastewater biological treatment targets. As the application of the yeast consortium for municipal wastewater treatment is a novel concept, this study focussed on characterisation and species identification to provide a foundation for future in-depth optimisation studies.
一项关于新型非无菌异养系统在去除市政污水中营养物质和有机物方面性能的研究已经完成。本研究中需要补充葡萄糖以消耗营养物质并提高生物量产量，因此，该系统可能对工业部门有吸引力，提供了一种可持续的方式来处理高有机负荷的污水或经过预处理的有机废物。微藻由于无法与快速繁殖的微生物竞争，生物量浓度较低，且营养物质去除效果较差。将微藻与酵母共培养导致微藻和酵母的产量均较低，主要原因是 pH 波动，但实现了极高的营养物质去除水平。市政污水中发现的野生酵母群落表现出最有前景的结果，持续实现高生物量浓度和营养物质去除率。在酵母培养物中，硝酸盐同化（一种在酵母中相对少见的特性）最大化了总氮的去除；而野生酵母群落的 Crabtree 阳性代谢表明同时实现污水处理和生物乙醇生产的可能性。因此，我们的研究表明，在达到污水生物处理目标的同时，可以产生有用的酵母生物质和生物乙醇。由于将酵母群落应用于市政污水处理是一个新颖的概念，本研究着重于表征和物种鉴定，为未来深入优化研究奠定基础。
一项关于新型非无菌异养系统在去除市政污水中营养物质和有机物方面性能的研究已经完成。本研究中需要补充葡萄糖以消耗营养物质并提高生物量产量，因此，该系统可能对工业部门有吸引力，提供了一种可持续的方式来处理高有机负荷的污水或经过预处理的有机废物。微藻由于无法与快速繁殖的微生物竞争，生物量浓度较低，且营养物质去除效果较差。将微藻与酵母共培养导致微藻和酵母的产量均较低，主要原因是 pH 波动，但实现了极高的营养物质去除水平。市政污水中发现的野生酵母群落表现出最有前景的结果，持续实现高生物量浓度和营养物质去除率。在酵母培养物中，硝酸盐同化（一种在酵母中相对少见的特性）最大化了总氮的去除；而野生酵母群落的 Crabtree 阳性代谢表明同时实现污水处理和生物乙醇生产的可能性。因此，我们的研究表明，在达到污水生物处理目标的同时，可以产生有用的酵母生物质和生物乙醇。由于将酵母群落应用于市政污水处理是一个新颖的概念，本研究着重于表征和物种鉴定，为未来深入优化研究奠定基础。
Declaration of Competing Interest
利益冲突声明
None. 无。
Acknowledgements 致谢
The authors acknowledge MEng Andrea Hernández García for her support and guidance at the Laboratorio de Ingeniería Ambiental Del Instituto de Ingeniería UNAM. The yeast initial morphological characterisation assistance provided by Dr Claudia Andrea Segal Kischinevzky and Ms Viviana Escobar Sánchez from the Laboratorio de Biología Molecular y Genómica de la Facultad de Ciencias UNAM is greatly appreciated. This work was supported by the British Council, UK (Grant no. 275897070) and CONACYT, Mexico (Grant no. 277914) under the Institutional Links Newton Fund Programme.
作者感谢工程硕士 Andrea Hernández García 在 UNAM 工程研究所环境工程实验室提供的支持和指导。特别感谢 UNAM 理学院分子生物学与基因组学实验室的 Claudia Andrea Segal Kischinevzky 博士和 Viviana Escobar Sánchez 女士在酵母初步形态特征分析方面的协助。本研究得到了英国文化协会（British Council, UK）（资助号：275897070）和墨西哥国家科学与技术委员会（CONACYT, Mexico）（资助号：277914）通过 Newton Fund 计划机构合作项目的资助。
作者感谢工程硕士 Andrea Hernández García 在 UNAM 工程研究所环境工程实验室提供的支持和指导。特别感谢 UNAM 理学院分子生物学与基因组学实验室的 Claudia Andrea Segal Kischinevzky 博士和 Viviana Escobar Sánchez 女士在酵母初步形态特征分析方面的协助。本研究得到了英国文化协会（British Council, UK）（资助号：275897070）和墨西哥国家科学与技术委员会（CONACYT, Mexico）（资助号：277914）通过 Newton Fund 计划机构合作项目的资助。
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