Comprehensive evaluation of a cost-effective method of culturing Chlorella pyrenoidosa with unsterilized piggery wastewater for biofuel production
利用未灭菌的养猪场废水培养小球藻 (Chlorella pyrenoidosa) 用于生物燃料生产的经济高效方法综合评估

Abstract  摘要

Background  背景

The utilization of Chlorella for the dual goals of biofuel production and wastewater nutrient removal is highly attractive. Moreover, this technology combined with flue gas (rich in CO₂) cleaning is considered to be an effective way of improving biofuel production. However, the sterilization of wastewater is an energy-consuming step. This study aimed to comprehensively evaluate a cost-effective method of culturing Chlorella pyrenoidosa in unsterilized piggery wastewater for biofuel production by sparging air or simulated flue gas, including algal biomass production, lipid production, nutrient removal rate and the mutual effects between algae and other microbes.
利用小球藻同时实现生物燃料生产和废水营养物去除的目标具有很大的吸引力。此外，将该技术与烟气（富含 CO₂）净化相结合，被认为是提高生物燃料产量的有效方法。然而，废水的杀菌处理是一个耗能的步骤。本研究旨在全面评估一种经济高效的方法，通过通入空气或模拟烟气，在未经杀菌处理的养猪场废水中培养奇异小球藻，用于生物燃料生产，包括藻类生物量产量、脂质产量、营养物去除率以及藻类与其他微生物之间的相互作用。

Results  结果

The average biomass productivity of C. pyrenoidosa reached 0.11 g L⁻¹ day⁻¹/0.15 g L⁻¹ day⁻¹ and the average lipid productivity reached 19.3 mg L⁻¹ day⁻¹/30.0 mg L⁻¹ day⁻¹ when sparging air or simulated flue gas, respectively. This method achieved fairish nutrient removal efficiency with respect to chemical oxygen demand (43.9%/55.1% when sparging air and simulated flue gas, respectively), ammonia (98.7%/100% when sparging air and simulated flue gas, respectively), total nitrogen (38.6%/51.9% when sparging air or simulated flue gas, respectively) and total phosphorus (42.8%/60.5% when sparging air or simulated flue gas, respectively). Culturing C. pyrenoidosa strongly influenced the microbial community in piggery wastewater. In particular, culturing C. pyrenoidosa enriched the abundance of the obligate parasite Vampirovibrionales, which can result in the death of Chlorella.
在通入空气或模拟烟道气的情况下，C. pyrenoidosa 的平均生物质生产率分别达到 0.11 g L⁻¹ day⁻¹和 0.15 g L⁻¹ day⁻¹，平均脂质生产率分别达到 19.3 mg L⁻¹ day⁻¹和 30.0 mg L⁻¹ day⁻¹。该方法在化学需氧量（分别为 43.9%和 55.1%）、氨氮（分别为 98.7%和 100%）、总氮（分别为 38.6%和 51.9%）以及总磷（分别为 42.8%和 60.5%）的去除效率方面表现出较好的效果。培养 C. pyrenoidosa 对养猪废水中的微生物群落具有显著影响，特别是培养 C. pyrenoidosa 会富集专性寄生菌 Vampirovibrionales 的数量，这可能导致小球藻的死亡。

Conclusion  结论

The study provided a comprehensive evaluation of culturing C. pyrenoidosa in unsterilized piggery wastewater for biofuel production. The results indicated that this cost-effective method is feasible but has considerable room for improving. More importantly, this study elucidated the mutual effects between algae and other microbes. In particular, a detrimental effect of the obligate parasite Vampirovibrionales on algal biomass and lipid production was found.
该研究对在未消毒的养猪场废水中培养小球藻用于生物燃料生产进行了全面评估。结果表明，这种具有成本效益的方法是可行的，但仍有很大的改进空间。更重要的是，研究阐明了藻类与其他微生物之间的相互作用，特别是专性寄生菌吸血弧菌目对藻类生物量和脂质产量的有害影响。

Background  背景

In the future, humans will face increasingly urgent challenges from the demand for energy. Unfortunately, fossil fuels are not sustainable energy resources. Therefore, the effective solution is to exploit renewable energy resources. At present, in view of their faster growth than other energy crops, microalgae are an ideal alternative to produce biodiesel [1, 2]. The growth of microalgae requires only sunlight, water, CO₂, and nutrients. It is well known that stock-farming wastewater, municipal wastewater and some industrial wastewaters are rich in nutrients, especially nitrogen (N) and phosphorus (P) [3]. Consequently, the utilization of microalgae for the dual goals of biomass production and wastewater purification is an eco-friendly industry with excellent prospects [2, 4, 5]. The utilization efficiency of CO₂ in microalgae can reach 20% [6]. Extra CO₂ supply is believed to be a promising approach for scaled-up algal biomass production [7]. To date, the eco-friendly biotechnology of using flue gas to cultivate microalgae has also been widely explored [8, 9].
未来，人类将面临日益紧迫的能源需求挑战。不幸的是，化石燃料并非可持续的能源资源。因此，有效的解决方案是开发可再生能源资源。目前，鉴于其比其他能源作物生长更快，微藻是生产生物柴油的理想替代品[1, 2]。微藻的生长只需要阳光、水、二氧化碳（CO₂）和营养物质。众所周知，畜牧废水、市政废水以及一些工业废水中富含营养物质，尤其是氮（N）和磷（P）[3]。因此，利用微藻实现生物质生产和废水净化双重目标是一项具有良好前景的环保产业[2, 4, 5]。微藻对 CO₂的利用效率可达 20%[6]。额外的 CO₂供应被认为是扩大藻类生物质生产的一个有前景的方法[7]。迄今为止，利用烟道气培养微藻的环保生物技术也已被广泛探索[8, 9]。

Chlorella with high carbohydrate or lipid content is an ideal material for biofuel production [10, 11]. Moreover, due to its high tolerance to soluble organic compounds, Chlorella is commonly used in wastewater treatment technology [12, 13]. In recent decades, the swine industry has developed rapidly in China, and the number of live swine has been ranked the highest in the world, resulting in serious environmental problems [14]. Piggery/swine wastewater hosts a complex community of microorganisms [15]. Bacterial infection represses the growth of some algae and simultaneously affects the algal cell density and lipid content [16]. Moreover, some bacteria can cause microalgae death by releasing soluble cellulose enzymes [17]. However, the detrimental effects of bacteria on Chlorella are unknown. To avoid such unknown detrimental effects, wastewater should be pretreated by sterilizing; however, this is a costly and energy-intensive process, which leads to bottlenecks in scaling up the cultivation of microalgae in piggery wastewaters [18, 19]. To date, there have been a number of studies regarding the technology of culturing Chlorella with sterilized piggery/swine wastewater [18,19,20,21,22,23,24,25,26,27,28,29,30,31]. However, little work has been reported on culturing Chlorella with unsterilized piggery/swine wastewater for biofuel production [32]. Therefore, the feasibility of culturing Chlorella with unsterilized piggery wastewater for biofuel production needs to be further demonstrated. More importantly, the relationship between bacteria and Chlorella needs to be clarified urgently. Consequently, this study aimed to comprehensively evaluate a cost-effective way of culturing Chlorella with unsterilized piggery wastewater for biofuel production under the condition of sparging air or simulated flue gas, including algal biomass production, lipid production and nutrient removal rate. More importantly, the mutual effects between algae and other microbes were also studied.
含有高碳水化合物或高脂质的小球藻是生产生物燃料的理想材料 [10, 11]。此外，由于其对可溶性有机化合物的高耐受性，小球藻常用于污水处理技术中 [12, 13]。近几十年来，中国生猪养殖业迅速发展，生猪饲养量位居世界第一，导致了严重的环境问题 [14]。养猪场污水中含有复杂的微生物群落 [15]。细菌感染会抑制某些藻类的生长，同时影响藻类的细胞密度和脂质含量 [16]。此外，一些细菌可以通过释放可溶性纤维素酶导致微藻死亡 [17]。然而，细菌对小球藻的有害影响尚不清楚。为避免这些未知的有害影响，污水应通过灭菌进行预处理；但这一过程成本高且耗能大，成为在养猪场污水中大规模培养微藻的瓶颈 [18, 19]。截至目前，已有多项关于使用灭菌养猪场污水培养小球藻的研究 [18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31]。然而，关于利用未灭菌养猪场污水培养小球藻以生产生物燃料的研究却少之又少 [32]。因此，亟需进一步论证利用未灭菌养猪场污水培养小球藻生产生物燃料的可行性。更重要的是，需要紧急澄清细菌与小球藻之间的关系。因此，本研究旨在全面评估一种经济高效的方法，在通入空气或模拟烟气的条件下，利用未灭菌养猪场污水培养小球藻生产生物燃料，包括藻类生物量生产、脂质生产和养分去除率。此外，还研究了藻类与其他微生物之间的相互作用。

Results and discussion  结果与讨论

Biomass and biofuel production of C. pyrenoidosa
**C. pyrenoidosa 的生物质和生物燃料生产**

According to the concentration range of nutrients reported in the previous literatures [18,19,20,21,22,23,24,25,26,27,28,29,30,31], the supernatant of piggery wastewater was diluted (1:4) with sterile water before used for culturing microalgae. The concentrations of COD, ammonium, total nitrogen, and total phosphorus in the diluted piggery wastewater were 327.3 mg L⁻¹, 11.6 mg L⁻¹, 33.7 mg L⁻¹ and 7.8 mg L⁻¹, respectively. After 10 days, the whole culturing process was finished. The growth potential of C. pyrenoidosa sparged with simulated flue gas was higher than that of C. pyrenoidosa sparged with air (Fig. 1a). The biomass concentration was 0.88/1.31 g L⁻¹, and the specific growth rate (μ) was 0.713/0.821 day⁻¹ when sparging air or simulated flue gas, respectively. Figure 1b shows the biomass productivity of C. pyrenoidosa in unsterilized piggery wastewater when sparging air or simulated flue gas. The average biomass productivity of C. pyrenoidosa sparged with simulated flue gas (0.15 g L⁻¹ day⁻¹) was higher than that of C. pyrenoidosa sparged with air (0.11 g L⁻¹ day⁻¹). Although the lipid content had no significant changes (Fig. 1c), the average lipid productivity when sparging simulated flue gas (30.0 mg L⁻¹ day⁻¹) was much higher than that under the condition of sparging air (19.3 mg L⁻¹ day⁻¹) due to the higher algal biomass productivity (Fig. 1d). Sparging flue gas into culture medium is an eco-effective way to increase the algal biomass, lipid content and production [8, 9]. Corresponding to the reported results, both algal biomass and lipid production were increased by sparging flue gas. Extra CO₂ supply can increase the lipid content of Chlorella, possibly because an elevated CO₂ concentration pushes cells to channel photosynthetic carbon precursors into fatty acid synthesis pathways, resulting in an increase in overall triacylglycerol generation [33]. However, the promoting effects of simulated flue gas on algal lipid content were weak in this study.
根据以往文献中报道的营养物质浓度范围 [18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31]，使用猪场废水上清液培养微藻之前，将其用无菌水稀释至 1:4。稀释后的猪场废水中化学需氧量（COD）、铵态氮、总氮和总磷的浓度分别为 327.3 mg/L、11.6 mg/L、33.7 mg/L 和 7.8 mg/L。整个培养过程持续了 10 天。通入模拟烟道气的 C. pyrenoidosa 的生长潜力高于通入空气的 C. pyrenoidosa（图 1a）。在通入空气或模拟烟道气时，生物质浓度分别为 0.88/1.31 g/L，特定生长速率（μ）分别为 0.713/0.821 天⁻¹。 图 1b 显示了在未灭菌的猪场废水中通入空气或模拟烟道气时 C. pyrenoidosa 的生物质生产能力。通入模拟烟道气的 C. pyrenoidosa 的平均生物质生产能力为 0.15 g/L·天，明显高于通入空气的平均生物质生产能力（0.11 g/L·天）。尽管脂质含量无显著变化（图 1c），但由于藻类的生物质生产能力更高，通入模拟烟道气时的平均脂质生产能力（30.0 mg/L·天）远高于通入空气时的脂质生产能力（19.3 mg/L·天）（图 1d）。 将烟道气通入培养基是一种生态高效的方法，可以提高藻类的生物质、脂质含量和生产效率 [8, 9]。与已有研究结果相符，通入烟道气可以同时提高藻类的生物质和脂质产量。额外的 CO₂ 供应可能通过提升细胞的 CO₂ 浓度，促进光合作用碳前体向脂肪酸合成途径转化，从而增加三酰基甘油的整体生成量 [33]。然而，在本研究中，模拟烟道气对藻类脂质含量的促进作用较弱。

Fig. 1  图 1

Algal biomass and lipid production of C. pyrenoidosa in unsterilized piggery wastewater when sparging air or simulated flue gas. The cultures were illuminated at 28 ± 0.5 °C under a 16-/8-h light/dark cycle with exposure to 45 μE m⁻² s⁻¹ provided by cool-white fluorescent lights. The microalgal cells were sampled every 24 h for growth determinations. PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. Data are presented as the mean ± standard deviation of the mean
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The biomass concentration, biomass productivity and lipid productivity of Chlorella in piggery wastewater, which varied in different studies, depended on the algal strain, nutrient components/concentration, ratio of C/N/P, pretreatment method, culture condition, etc. [18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]. Therefore, it is insufficient to evaluate a technology just based on biomass concentration, biomass productivity and lipid productivity. In our study, these parameters had considerable room for improving by optimizing the nutrient components/concentration, nutrient ratio (C/N/P), illumination intensity, aeration mode and so on. Sterilization is indeed a costly and energy-intensive process, which leads to bottlenecks in scaling up the cultivation of microalgae in piggery wastewaters [18, 19]. Consequently, the method of culturing Chlorella with unsterilized piggery wastewater for biofuel production should be regarded as a sustainable and cost-effective technology.
在不同研究中，养猪场废水中小球藻的生物量浓度、生物量生产率和脂质生产率有所不同，这取决于藻株、营养成分/浓度、C/N/P 比例、预处理方法、培养条件等因素[18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32]。因此，仅根据生物量浓度、生物量生产率和脂质生产率来评估一项技术是不充分的。在我们的研究中，通过优化营养成分/浓度、营养比例（C/N/P）、光照强度、通气方式等参数，这些指标还有很大的提升空间。灭菌确实是一个成本高且耗能的过程，这成为在养猪场废水中规模化培养微藻的瓶颈[18, 19]。因此，利用未灭菌的养猪场废水培养小球藻生产生物燃料的方法应被视为一种可持续且具有成本效益的技术。

Nutrient removal efficiency
营养去除效率

The nutrient removal efficiencies of culturing C. pyrenoidosa in unsterilized piggery wastewater when sparging air or simulated flue gas were studied (Fig. 2). The concentration of COD experienced an obvious decrease when culturing C. pyrenoidosa in this study. The removal rate of COD was 43.9%/55.1% when sparging air or simulated flue gas, respectively. When Chlorella was cultured in piggery/swine wastewater, the COD removal rate varied in different studies [18,19,20,21,22,23,24,25,26,27,28,29,30,31,32, 34]. A COD removal rate achieved 99% by reducing ammonia concentration and optimizing C/N ratio (25:1) with culturing C. vulgaris after 7-day cultivation in sterilized piggery wastewater, which was the maximum in the current literatures [20]. In this study, the ammonium removal rate of C. pyrenoidosa was 98.7% when sparging air and 100% when sparging simulated flue gas, while it fluctuated between 70 and 100% in the reported results [18,19,20,21,22,23,24,25,26,27,28,29,30,31,32, 34]. The high removal efficiency was due to ammonium being the preferred nitrogen source for most microalgae [35]. In addition, even when C. pyrenoidosa was not cultured, the decrease in ammonium concentration was also obvious when sparging air. This should be attributed to the ammoxidation—a biochemical process needing oxygen. Sparging air promoted this biochemical process, resulting in a significant reduction in ammonium. The highest removal rate of TN was also reported by Zheng et al. [20]. The highest removal rate of TP in sterilized piggery wastewater was 98.17% when culturing C. zofingiensis [19]. In this study, the TN removal rates of C. pyrenoidosa were 38.6% when sparging air and 51.9% when sparging simulated flue gas, while the removal rates of TP were 42.8% when sparging air and 60.5% when sparging simulated flue gas.
研究了在未灭菌的养猪废水中培养小球藻（C. pyrenoidosa）时通入空气或模拟烟道气对营养物质去除效率的影响（图 2）。研究中，化学需氧量（COD）的浓度显著降低。在通入空气或模拟烟道气的条件下，COD 的去除率分别为 43.9%和 55.1%。在不同研究中，当小球藻在养猪废水中培养时，COD 的去除率存在差异[18-34]。在灭菌的养猪废水中，通过减少氨浓度和优化碳氮比（25:1），培养普通小球藻（C. vulgaris）7 天后，COD 去除率最高达到 99%，这是当前文献中报道的最大值[20]。 本研究中，当通入空气时，小球藻对氨氮的去除率为 98.7%；通入模拟烟道气时，氨氮去除率为 100%，而在已有研究结果中，该去除率波动在 70%-100%之间[18-34]。高去除效率的原因是氨氮是大多数微藻的优先氮源[35]。此外，即使不培养小球藻，在通入空气时氨氮浓度的减少也非常明显，这应该归因于需氧条件下的氨氧化生化过程。通入空气促进了这一生化过程，从而显著降低了氨氮浓度。最高的总氮（TN）去除率也由 Zheng 等人报道[20]。在灭菌的养猪废水中，培养着色小球藻（C. zofingiensis）时，总磷（TP）的最高去除率为 98.17%[19]。本研究中，小球藻对 TN 的去除率为 38.6%（通入空气）和 51.9%（通入模拟烟道气）；对 TP 的去除率为 42.8%（通入空气）和 60.5%（通入模拟烟道气）。

Fig. 2  图 2

Chemical oxygen demand (COD), ammonium (NH₄⁺-N), total nitrogen (TN) and total phosphate (TP) removal rates. CA means sparging air, CC sparging simulated flue gas, PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. Data are presented as the mean ± standard deviation of the mean. **Indicates that there was an extremely significant difference with P < 0.01
化学需氧量（COD）、氨氮（NH₄⁺-N）、总氮（TN）和总磷（TP）去除率。CA 表示通入空气，CC 表示通入模拟烟气，PA 表示在通入空气条件下培养小球藻（C. pyrenoidosa），PC 表示在通入模拟烟气条件下培养小球藻（C. pyrenoidosa）。数据以平均值±标准偏差表示。**表示差异极显著，P < 0.01。

Effects of culturing C. pyrenoidosa on bacterial abundance and community
**培养小球藻（C. pyrenoidosa）对细菌丰度和群落的影响**

Figure 3 shows the effects of culturing C. pyrenoidosa on bacterial abundance in unsterilized piggery wastewater. When sparging air, culturing C. pyrenoidosa suppressed the bacterial abundance significantly: the number of 16S rRNA gene copies decreased from 1.3 × 10⁸ copies mL⁻¹ (without culturing C. pyrenoidosa) to 3.2 × 10⁵ copies mL⁻¹ (culturing C. pyrenoidosa). However, when sparging simulated flue gas, culturing C. pyrenoidosa had no effect on bacterial abundance.
图 3 展示了在未经灭菌的养猪场废水中培养小球藻（C. pyrenoidosa）对细菌丰度的影响。在通入空气的情况下，培养小球藻显著抑制了细菌的丰度：16S rRNA 基因拷贝数从 **1.3 × 10⁰ 拷贝/mL**（未培养小球藻时）下降到 **3.2 × 10² 拷贝/mL**（培养小球藻时）。然而，在通入模拟烟气的情况下，培养小球藻对细菌丰度没有影响。

Fig. 3  图 3

The absolute bacterial abundance based on 16S rRNA copies in unsterilized piggery wastewater. CA means sparging air, CC means sparging simulated flue gas, PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. Data are presented as the mean ± standard deviation of the mean. The different letters indicate that there was a significant difference with P < 0.05
基于 16S rRNA 拷贝的未灭菌养猪场废水中细菌绝对丰度。CA 表示通入空气，CC 表示通入模拟烟气，PA 表示通入空气培养 C. pyrenoidosa，PC 表示通入模拟烟气培养 C. pyrenoidosa。数据以平均值±标准偏差表示，不同字母表示存在显著差异（P < 0.05）。

The analysis of the bacterial community provided deep insights into the mutual effects between C. pyrenoidosa and other microbes. The high-throughput sequencing of 16S rRNA V4 region amplicons yielded 1,073,927 raw reads. After filtering low-quality reads and trimming the adapters, barcodes and primers, there were 1,012,135 valid reads (average length 253 bp). A total of 3185 operational taxonomic units (OTUs) (97% sequence similarity) were clustered. The bacterial abundance was significantly suppressed by culturing C. pyrenoidosa under sparging air (Fig. 3), whereas the bacterial diversity was increased (Fig. 4). Under sparging simulated flue gas, culturing C. pyrenoidosa decreased the microbial diversity (Fig. 4).
对细菌群落的分析深入揭示了小球藻（C. pyrenoidosa）与其他微生物之间的相互作用。对 16S rRNA V4 区扩增子进行高通量测序，共获得 1,073,927 条原始序列。经过过滤低质量序列并去除接头、条形码和引物后，得到 1,012,135 条有效序列（平均长度为 253 bp）。以 97%的序列相似性聚类，共获得 3185 个操作分类单元（OTUs）。在通入空气的条件下培养小球藻显著抑制了细菌的丰度（图 3），但增加了细菌的多样性（图 4）。而在通入模拟烟道气的条件下培养小球藻则降低了微生物的多样性（图 4）。

Fig. 4  图 4

The boxplots of alpha indices (Chao1 and ACE). CA means sparging air, CC sparging simulated flue gas, PA means culturing C. pyrenoidosa with sparging air, and PC means culturing C. pyrenoidosa with sparging simulated flue gas. *Indicates that there was a significant difference with P < 0.05. The statistical test used to compare the indices was the Wilcoxon signed-rank test
α指数（Chao1 和 ACE）的箱线图。CA 表示通入空气，CC 表示通入模拟烟气，PA 表示在通入空气条件下培养小球藻，PC 表示在通入模拟烟气条件下培养小球藻。*表示显著性差异，P < 0.05。用于比较指数的统计检验方法为 Wilcoxon 符号秩检验。

LEfSe clearly indicated the effects of culturing C. pyrenoidosa on the bacteria (Figs. 5 and 6). Dogs et al. [36] found that Rhodobacteraceae was the predominant family constituting 23% of the epibacterial community of the marine brown algae Fucus spiralis and showed physiological adaptation to an epiphytic lifestyle. In this study, Rhodobacteraceae with a relative abundance of 6.8% was also the dominant family in samples culturing C. pyrenoidosa when sparging air. It has been reported that the family Rhodobacteraceae is deeply involved in sulfur and carbon biogeochemical cycling [37]. Vampirovibrionales, commonly found in the human gut and groundwater, belongs to a new phylum related to Cyanobacteria. The members of Vampirovibrionales are obligate parasites that attach to the cell wall of green alga of Chlorella [38], resulting in the death of Chlorella. In this study, Vampirovibrionales were the major bacteria, constituting 0.7–6.7% of the bacterial community in the detected samples. It is noteworthy that the Vampirovibrionales were significantly enriched by culturing C. pyrenoidosa. Under the condition of sparging air, the abundance of Vampirovibrionales when culturing C. pyrenoidosa was 7.6 times as high as that without culturing C. pyrenoidosa. Under the condition of sparging simulated flue gas, the abundance of Vampirovibrionales with culturing C. pyrenoidosa was 2.7 times as high as that without culturing C. pyrenoidosa. The results indicated that C. pyrenoidosa suffered from infection by Vampirovibrionales, and this greatly impeded the increase in algal biomass. Pedobacter glucosidilyticus was also enriched by culturing C. pyrenoidosa, whereas Kerstersia gyiorum, MNG7 and Saprospiraceae were suppressed. K. gyiorum is a pathogenic member of the family Alcaligenaceae and is commonly isolated from leg wounds, chronic ear infections, human feces, sputum, and even bronchoalveolar lavage fluids and the urinary tract [39,40,41]. The suppression of pathogenic microbes by C. pyrenoidosa might contribute to a decrease in the risk to public health. The Saprospiraceae, a family within the order Sphingobacteriales, have a demonstrated ability for the hydrolysis and utilization of some complex organic sources [42]. Under the condition of sparging simulated flue gas (Fig. 5), Comamonadaceae, Draconibacteriaceae, Sediminibacterium, Sterolibacterium, and K. gyiorum were significantly suppressed by C. pyrenoidosa. However, the bacteria of Alphaproteobacteria, Melainabacteria, Vibrio, and Thermomonas fusca were enriched by culturing C. pyrenoidosa. Sterolibacterium, commonly found in anoxic environments, can reduce nitrate to dinitrogen [43].
LEfSe 清晰地表明培养裂壶藻（*C. pyrenoidosa*）对细菌群落的影响（图 5 和图 6）。Dogs 等人[36]发现，红杆菌科（*Rhodobacteraceae*）是构成海洋褐藻螺旋海带（*Fucus spiralis*）表面细菌群的主要家族，占 23%，并表现出对附生生活方式的生理适应。在本研究中，当通入空气时，样本中红杆菌科的相对丰度为 6.8%，也是培养裂壶藻时的主要家族。据报道，红杆菌科在硫和碳的生物地球化学循环中发挥着重要作用[37]。吸血菌目（*Vampirovibrionales*）通常存在于人类肠道和地下水中，属于一个与蓝藻相关的新门类。吸血菌目成员是专性寄生菌，附着在小球藻（*Chlorella*）的细胞壁上[38]，导致小球藻死亡。在本研究中，吸血菌目是主要细菌，在检测的样本中占 0.7%–6.7%的细菌群落比例。值得注意的是，培养裂壶藻显著富集了吸血菌目。在通入空气的条件下，培养裂壶藻时吸血菌目的丰度是未培养裂壶藻时的 7.6 倍。在通入模拟烟气条件下，培养裂壶藻时吸血菌目的丰度是未培养裂壶藻时的 2.7 倍。结果表明，裂壶藻受到吸血菌目的感染，这极大地阻碍了藻类生物量的增加。培养裂壶藻还富集了葡糖水解微杆菌（*Pedobacter glucosidilyticus*），而抑制了吉奥鲁姆克氏菌（*Kerstersia gyiorum*）、MNG7 和腐螺菌科（*Saprospiraceae*）。克氏菌（*K. gyiorum*）是碱杆菌科（*Alcaligenaceae*）的致病菌，通常从腿部伤口、慢性耳部感染、人类粪便、痰液，甚至支气管肺泡灌洗液和尿道中分离[39, 40, 41]。裂壶藻抑制致病微生物可能有助于降低公共健康风险。腐螺菌科是球形杆菌目（*Sphingobacteriales*）中的一个家族，已被证明具有水解和利用某些复杂有机物的能力[42]。在通入模拟烟气的条件下（图 5），裂壶藻显著抑制了庞大单胞菌科（*Comamonadaceae*）、龙杆菌科（*Draconibacteriaceae*）、沉积杆菌（*Sediminibacterium*）、固醇杆菌（*Sterolibacterium*）和克氏菌（*K. gyiorum*）。然而，裂壶藻培养中富集了α-变形菌纲（*Alphaproteobacteria*）、黑藻菌纲（*Melainabacteria*）、弧菌（*Vibrio*）和耐热单胞菌（*Thermomonas fusca*）。固醇杆菌通常存在于缺氧环境中，可以将硝酸盐还原为氮气[43]。

Fig. 5  图 5

LEfSe analysis identified the most differentially abundant taxa between CA and PA. The taxonomic cladogram was obtained from LEfSe analysis of 16S rRNA sequences; only taxa meeting an LDA significance threshold of 4.0 are shown. Small circles and shading with different colors in the diagram represent the abundance of those taxa in the respective group. Yellow circles represent nonsignificant differences in abundance between CA and PA for that particular taxonomic group. The brightness of each dot is proportional to its effect size. Taxa enriched in PA are shown with a positive LDA score (green) and taxa enriched in CA have a negative score (red). CA means sparging air; PA means culturing C. pyrenoidosa with sparging air
LEfSe 分析识别了 CA 和 PA 之间差异最显著的分类单元。通过对 16S rRNA 序列进行 LEfSe 分析获得了分类学进化树；仅显示满足 LDA 显著性阈值 4.0 的分类单元。图中不同颜色的小圆圈和阴影表示这些分类单元在各自组中的丰度。黄色圆圈表示该特定分类单元在 CA 和 PA 之间的丰度差异不显著。每个点的亮度与其效应大小成正比。丰度在 PA 中增加的分类单元显示为正 LDA 得分（绿色），而丰度在 CA 中增加的分类单元显示为负得分（红色）。CA 表示通入空气；PA 表示在通入空气条件下培养 C. pyrenoidosa。

Fig. 6  图 6

LEfSe analysis identified the most differentially abundant taxa between CC and PC. The taxonomic cladogram was obtained from LEfSe analysis of 16S rRNA sequences; only taxa meeting an LDA significance threshold of 4.0 are shown. Small circles and shading with different colors in the diagram represent the abundance of those taxa in the respective group. Yellow circles represent nonsignificant differences in abundance between CC and PC for that particular taxonomic group. The brightness of each dot is proportional to its effect size. Taxa enriched in PC are shown with a positive LDA score (green), and taxa enriched in CC have a negative score (red). CC means sparging simulated flue gas; PC means culturing C. pyrenoidosa with sparging simulated flue gas
LEfSe 分析鉴定了 CC 和 PC 之间差异最显著的分类单元。分类系统树图基于 16S rRNA 序列的 LEfSe 分析获得；仅显示满足 LDA 显著性阈值 4.0 的分类单元。图中的小圆圈和不同颜色的阴影表示这些分类单元在各组中的丰度。黄色圆圈表示该分类单元在 CC 和 PC 之间的丰度差异不显著。每个点的亮度与其效应大小成正比。在 PC 中富集的分类单元显示为正的 LDA 得分（绿色），而在 CC 中富集的分类单元则显示为负的 LDA 得分（红色）。CC 表示通入模拟烟道气的培养条件；PC 表示在培养条件下通入模拟烟道气对 C. pyrenoidosa 的培养。

Clearly, simulated flue gas and culturing C. pyrenoidosa both played key roles in structuring the bacterial community. In fact, there are other non-negligible factors that might influence the bacterial community: (1) Algae can excrete a variety of organic compounds, such as carbohydrates, lipopolysaccharides, organohalogens, amino acids and peptides, which are available to many bacteria [44]. In this study, some organic matter originating from C. pyrenoidosa could be utilized by specific bacteria during cultivation. However, some studies have indicated that some organic matter of Chlorella has antibacterial activity against specific bacteria [45]. Therefore, it is probable that some bacteria in piggery wastewater were inhibited by culturing C. pyrenoidosa. (2) The growth of C. pyrenoidosa had little effect on pH when sparging simulated flue gas in this study, but the pH (> 8.0) was increased by C. pyrenoidosa when sparging air (Additional file 1: Fig. S1). When phytoplankton grows in excessive abundance, photosynthesis by algae during daylight releases oxygen and removes carbon dioxide from the water, resulting in an increase in pH [46, 47]. Consequently, pH influences the bacterial community. (3) Nutrient competition can also influence the relationship between microalgae and bacteria [48, 49]. In this study, the concentrations of ammonium, TN and TP decreased due to culturing C. pyrenoidosa, which might also lead to changes in the bacterial community.
显然，模拟烟气和培养小球藻（C. pyrenoidosa）在构建细菌群落方面都起到了关键作用。实际上，还有其他不可忽视的因素可能影响细菌群落： (1) 藻类可以分泌多种有机化合物，例如碳水化合物、脂多糖、有机卤化物、氨基酸和肽类，这些都可被许多细菌利用 [44]。在本研究中，一些来源于小球藻的有机物在培养过程中可能被特定细菌利用。然而，一些研究表明，小球藻的某些有机物对特定细菌具有抗菌活性 [45]。因此，有可能养殖废水中的某些细菌因培养小球藻而受到抑制。 (2) 在本研究中，用模拟烟气曝气时，小球藻的生长对 pH 的影响很小，但在用空气曝气时，小球藻将 pH 提高到 8.0 以上（附加文件 1：图 S1）。当浮游植物过度繁殖时，藻类在白天通过光合作用释放氧气并消耗水中的二氧化碳，从而导致 pH 值升高 [46, 47]。因此，pH 会影响细菌群落。 (3) 营养竞争也会影响微藻与细菌之间的关系 [48, 49]。在本研究中，由于培养小球藻，氨氮（NH₄⁺）、总氮（TN）和总磷（TP）的浓度下降，这也可能导致细菌群落的变化。

The most noteworthy result was that the obligate parasites Vampirovibrionales were significantly enriched by culturing C. pyrenoidosa. The bacterium has very specific requirements for growth—it seems to grow only by attachment to the cell wall of intact Chlorella cells and consuming their cytoplasmic contents [38, 50]. Although it needs to be further clarified whether the obligate parasites Vampirovibrionales are commonly found in other wastewaters, this result emphasizes the need to adequately consider these obligate parasites when using unsterilized wastewater for culturing Chlorella. In other words, the obligate parasite Vampirovibrionales in this study was a restrictive factor in algal growth, lipid accumulation and nutrient removal. More importantly, this result indicates that the selection of algal strain must be carefully performed.
最值得注意的结果是，专性寄生菌吸血鬼菌目（Vampirovibrionales）在培养小球藻（C. pyrenoidosa）时显著富集。该细菌对生长有非常特定的要求——它似乎只能通过附着在完整小球藻细胞的细胞壁上并消耗其细胞质内容物来生长[38, 50]。尽管尚需进一步明确专性寄生菌吸血鬼菌目是否常见于其他废水中，但这一结果强调了在使用未灭菌废水培养小球藻时，需要充分考虑这些专性寄生菌。换句话说，本研究中的专性寄生菌吸血鬼菌目是藻类生长、脂质积累和营养去除的限制性因素。更重要的是，这一结果表明必须谨慎选择藻类菌株。

Conclusion  结论

In this study, we comprehensively evaluated a cost-effective method of using unsterilized piggery wastewater for biofuel production by culturing Chlorella. This method achieved moderate algal biomass productivity, lipid productivity and fairish nutrient removal efficiency. Moreover, our results indicated that culturing C. pyrenoidosa strongly influenced the microbial community in piggery wastewater. In particular, a detrimental effect of the obligate parasite Vampirovibrionales on algal biomass and lipid production was found.
在本研究中，我们全面评估了一种利用未消毒的养猪场废水培养小球藻生产生物燃料的经济高效方法。该方法实现了中等水平的藻类生物量生产率、脂质生产率以及较为适中的养分去除效率。此外，我们的结果表明，培养裂殖壶藻（C. pyrenoidosa）对养猪场废水中的微生物群落有显著影响。特别是，发现专性寄生菌吸血鬼弧菌目（Vampirovibrionales）对藻类生物量和脂质生产具有不利影响。

Methods  方法

Piggery wastewater used as culture media
**以养猪场废水作为培养基**

The piggery wastewater used in this study was from a local pig farm, was directly discharged and was stored in a cement pond. The collected wastewater was allowed to settle for 1 day to precipitate. The supernatant was diluted (1:4) with sterile water before being used for culturing microalgae. The concentrations of COD, ammonium, total nitrogen, and total phosphorus in the piggery wastewater were determined following the protocols described previously [51], and the parameters of the original piggery wastewater are shown in Additional file 2: Table S1.
本研究中使用的养猪场废水来源于当地的一家养猪场，废水直接排放并储存在一个水泥池中。收集的废水静置 1 天以进行沉淀。上清液用无菌水按照 1:4 的比例稀释后，用于培养微藻。废水中化学需氧量（COD）、氨氮、总氮和总磷的浓度按照之前描述的实验方法测定 [51]，原始养猪场废水的具体参数见附加文件 2：表 S1。

Algal strain and culture conditions
**藻株及培养条件**

C. pyrenoidosa, a species of Chlorella, can tolerate a high concentration of soluble organic compounds and effectively utilize a variety of organic carbon sources in wastewater [52, 53]. Therefore, C. pyrenoidosa was selected as a target strain. The green algae C. pyrenoidosa was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (FACHB-10), and grown in BBM medium containing the following composition (per liter): 0.25 g NaNO₃, 0.075 g K₂HPO₄, 0.075 g MgSO₄·7H₂O, 0.025 g CaCl₂·2H₂O, 0.175 g KH₂PO₄, 0.025 g NaCl, 0.75 mg Na₂-EDTA, 0.097 mg FeCl₃·6H₂O, 1 mg vitamin B₁, 0.25 μg biotin, 0.15 μg vitamin B₁₂, 0.041 mg MnCl₂·4H₂O, 0.005 mg ZnCl₂·7H₂O, 0.004 mg Na₂MoO₄·2H₂O and 0.002 mg CoCl₂·6H₂O. The algal cells were axenically grown at 28 ± 0.5 °C under a 16-/8-h light/dark cycle with exposure to 45 μE m⁻² s⁻¹ provided by cool-white fluorescent lights. The cool-white fluorescent lights were 0.2 m above the culture flask. After adjusting the pH to 7.0, 500 mL of the pretreated piggery wastewater was placed in a 2000-mL conical flask. C. pyrenoidosa in the linear growth phase was used as the inoculum. The initial inoculation density was 2 × 10⁶ cells mL⁻¹. The culture medium without mechanical oscillation was sparged with sterilized air or simulated flue gas (CO₂ 20%, N₂ 80%) at a flow rate of 0.5 L min⁻¹. The experiments were divided into four groups: sparging air (CA), sparging air with culturing C. pyrenoidosa (PA), sparging simulated flue gas (CC) and sparging CO₂ with culturing C. pyrenoidosa (PC). All experiments were conducted in triplicate.
C. pyrenoidosa（一种小球藻物种）能够耐受高浓度的可溶性有机化合物，并能有效利用废水中的多种有机碳源 [52, 53]。因此，C. pyrenoidosa 被选为目标菌株。这种绿色藻类 C. pyrenoidosa 来自中国科学院水生生物研究所（FACHB-10），并在含有以下成分的 BBM 培养基中培养（每升）：0.25 g NaNO₃、0.075 g K₂HPO₄、0.075 g MgSO₄·7H₂O、0.025 g CaCl₂·2H₂O、0.175 g KH₂PO₄、0.025 g NaCl、0.75 mg Na-EDTA、0.097 mg FeCl₃·6H₂O、1 mg 维生素 B₁₂、0.25 μg 生物素、0.15 μg 维生素 B₆、0.041 mg MnCl₂·4H₂O、0.005 mg ZnCl₂·7H₂O、0.004 mg Na₂MoO₄·2H₂O 和 0.002 mg CoCl₂·6H₂O。藻细胞在 28 ± 0.5 °C 下无菌培养，光暗周期为 16 小时光照/8 小时黑暗，光照强度为 45 μE·m⁻²·s⁻¹，由冷白色荧光灯提供，灯距培养瓶 0.2 m。在将 pH 调至 7.0 后，将 500 mL 预处理过的养猪废水置于 2000 mL 的锥形瓶中。处于线性生长期的 C. pyrenoidosa 用作接种物，初始接种密度为 2 × 10⁶ 个细胞/mL。培养基在没有机械振荡的情况下，通过灭菌空气或模拟烟气（CO₂ 20%，N₂ 80%）以 0.5 L/min 的流速充气。实验分为四组：充空气（CA）、充空气培养 C. pyrenoidosa（PA）、充模拟烟气（CC）和充 CO₂ 培养 C. pyrenoidosa（PC）。所有实验均进行三次重复。

Growth of C. pyrenoidosa
小球藻（C. pyrenoidosa）的生长

The growth of C. pyrenoidosa was determined by measuring the total chlorophyll concentration (∑C) using a spectrophotometric method [18, 54]. The biomass concentration (dry weight of cell powder (DCW) in culture medium, g L⁻¹) in the piggery wastewater was estimated by an equation that employs the total chlorophyll (∑C):
**C. pyrenoidosa 的生长通过使用分光光度法测量总叶绿素浓度（∑C）来确定 [18, 54]。在猪场废水中，生物量浓度（培养基中细胞粉末的干重 (DCW)，g/L）通过一个利用总叶绿素（∑C）的公式进行估算：**

$$\text{DCW }\left({\text{g L}}^{-1}\right)=0.1084\sum C,R^2=0.9562$$

(1)

The specific growth rate (μ) was calculated by fitting the total chlorophyll in the exponential phase of algal growth, which was measured by the following formula:
**特定生长速率（μ）通过拟合藻类生长指数阶段的总叶绿素数据计算，具体采用以下公式：**

$$\mu \left({\text{day}}^{-1}\right)=\left(\ln \sum C-\ln \sum C_0\right)/\phantom{\left(\ln \sum C-\ln \sum C_0\right)t}t,$$

(2)

where t (day) is the time between two measurements and ∑C and ∑C₀ (mg L⁻¹) are the total chlorophyll concentrations at the start and end of the exponential phase, respectively. The biomass productivity (P) was calculated according to the following formula [7]:
其中，t（天）是两次测量之间的时间，∑C 和 ∑C₀（mg/L）分别表示指数生长期开始和结束时的总叶绿素浓度。生物量生产率（P）按照以下公式计算 [7]：

$$P=\left(d{w}_i-d{w}_o\right)/\left(t_i-t_0\right),$$

(3)

where dw_i and dw_o are dry biomass (g L⁻¹) at time t_i and t₀ (initial time), respectively.
其中，dw₀ 和 dw₁ 分别表示在时间 t₃ 和 t₄（初始时间）的干生物量（g/L²）。

Determination of lipid, protein and carbohydrate content and productivity
脂质、蛋白质和碳水化合物含量及生产率的测定

The biochemical composition of algae was determined by Fourier transform infrared (FTIR) spectrometry. The FTIR analysis was performed as previously described by Zhang et al. [53]. Briefly, cell pellets centrifuged at 8000g for 10 min were washed twice with deionized water. Deionized water was used to resuspend the cell pellets at a concentration of approximately 1.0 mg mL⁻¹ (dry weight). A vacuum drying oven was used to dry a total of 200-μL suspension, which was dropped on a KRS-5 window (30 × 5 mm) at 40 °C. The transmittance spectra were collected between 400 and 4000 cm ⁻¹ at a resolution of 4 cm⁻¹ with 32 scans on an FTIR spectrometer (NEXUS 870, Thermo Nicolet, USA). The data were processed with OMNIC 6.0 software. The spectrum baseline was corrected by a rubber-band method using 64 baseline points with the exclusion of CO₂ bands.
藻类的生化成分通过傅里叶变换红外光谱（FTIR）法测定。FTIR 分析按照 Zhang 等人[53]的方法进行。简而言之，将以 8000g 离心 10 分钟后的细胞沉淀用去离子水清洗两次，并用去离子水以约 1.0 mg/mL（干重）的浓度重悬。将 200 μL 的悬液滴在 KRS-5 窗口（30 × 5 mm）上，在 40°C 下使用真空干燥箱干燥。透射光谱在 400 至 4000 cm⁻¹的范围内，以 4 cm⁻¹的分辨率通过 FTIR 光谱仪（NEXUS 870，Thermo Nicolet，美国）扫描 32 次收集。数据通过 OMNIC 6.0 软件处理。光谱基线采用橡皮带法校正，使用 64 个基线点，并排除了 CO₃波段的影响。

The characteristic peak areas of lipids (A_L), proteins (A_P) and carbohydrates (A_C) were calculated by integration. The weights (mg) of lipids (W_L), proteins (W_P) and carbohydrates (W_C) were calculated according to the following formulas [55]:
脂类特征峰面积（A _L ）、蛋白质特征峰面积（A _P ）和碳水化合物特征峰面积（A _C ）通过积分计算得出。脂类（W _L ）、蛋白质（W _P ）和碳水化合物（W _C ）的重量（毫克）根据以下公式计算 [55]：

$$A_L=-2.30+78.96\times W_L$$

(4)

$$A_p=-0.27+12.72\times W_p$$

(5)

$$A_p=-0.27+12.72\times W_p$$

(6)

Assuming that the algal cells consisted of only lipids, proteins and carbohydrates, the contents (%) of lipids (C_L), proteins (C_P) and carbohydrates (C_C) were calculated with the following formulas [56]:
假设藻类细胞仅由脂类、蛋白质和碳水化合物组成，脂类（C _L ）、蛋白质（C _P ）和碳水化合物（C _C ）的含量（%）通过以下公式计算 [56]：

$$C_L={\text{W}}_{\mathrm{L}}/({\text{W}}_{\mathrm{L}}+{\text{W}}_{\mathrm{P}}+{\text{W}}_{\mathrm{C}})\times 100$$

(7)

$$C_P={\text{W}}_{\mathrm{P}}/({\text{W}}_{\mathrm{L}}+{\text{W}}_{\mathrm{P}}+{\text{W}}_{\mathrm{C}})\times 100$$

(8)

$$C_C={\text{W}}_{\mathrm{C}}/({\text{W}}_{\mathrm{L}}+{\text{W}}_{\mathrm{P}}+{\text{W}}_{\mathrm{C}})\times 100$$

(9)

The lipid productivity (P_L) was calculated according to the following formula:
脂质生产率（P _L ）根据以下公式计算：

$$P_L=(d{w}_i-d{w}_o)\times C_L/((t_i-t_0)\times 100),$$

(10)

where dw_i and dw_o are the dry biomass (g L⁻¹) at times t_i and t₀ (initial time), respectively. C_L is the lipid content (%).
其中，dw _i 和 dw _o 分别是时间 t _i 和 t ₀ （初始时间）的干生物量（g L ⁻¹ ）。C _L 是脂质含量（%）。

Sampling and nutrient analysis
采样与营养分析

A volume of 5-mL microalgae suspension was collected every day from each conical flask in a clean bench for nutrient analysis starting from inoculation. The samples were first centrifuged at 5000 rpm for 10 min, after which the supernatants were filtered using a 0.22-μm nylon membrane filter. Then, the filtrates were appropriately diluted and analyzed for ammonia, total nitrogen, and total phosphate following the Hach DR 2700 Spectrophotometer Manual. The nutrient removal rate was obtained using the following expression [19]:
从接种开始，每天从每个锥形瓶中在洁净台上取 5 毫升微藻悬浮液用于营养分析。样品首先以 5000 rpm 离心 10 分钟，然后将上清液通过 0.22 μm 尼龙膜过滤器过滤。随后，将滤液适当稀释后，按照 Hach DR 2700 分光光度计手册分析其中的氨、总氮和总磷。营养去除率根据以下公式计算 [19]：

$$NutrientremovalrateW\mathrm{\%}=100\mathrm{\%}\times (C_0-C_i)/C_0,$$

(11)

where C_o and C_i are defined as the mean nutrient concentrations at the initial time t₀ and time t_i, respectively.
其中，C _o 和 C _i 分别被定义为初始时间 t ₀ 和时间 t _i 时的平均养分浓度。

DNA extraction and sequencing library construction
**DNA 提取和测序文库构建**

After the C. pyrenoidosa grew for 10 days, the medium was oscillated at a speed of 100 r min⁻¹, and then 0.05-L samples from each flask were filtered with 0.22-μm filter membranes using a filtration apparatus. The obtained membranes were stored at − 80 °C until DNA extraction. Before DNA extraction, all the filter membranes were cut into pieces with sterile scissors. DNA extraction was performed using an E.Z.N.A. Water DNA Kit (OMEGA Bio-Tek Inc., USA) according to the manufacturer’s instructions. The extracted DNA was stored in a freezer at − 80 °C prior to downstream analysis. The 16S rRNA amplicons were amplified by primer pair 515F/806R (515F: 5′-NNNNNNNNGTGTGCCAGCMGCCGCGGTAA-3′, 806R: 5′-GGACTACHVGGGTWTCTAAT-3′) targeting the V4 hypervariable region of 16S rRNA genes [57]. The high-throughput sequencing of 16S rRNA amplicons was performed on the Illumina MiSeq platform at Novogene Bioinformatics Company (Beijing, China).
在小球藻 (*C. pyrenoidosa*) 生长 10 天后，将培养基以 100 r/min 的速度振荡，然后使用过滤装置通过 0.22 μm 过滤膜过滤每个培养瓶中的 0.05 L 样品。过滤后的膜被存储在-80°C 的条件下，直至进行 DNA 提取。在进行 DNA 提取之前，所有过滤膜均使用无菌剪刀剪成小块。DNA 提取使用 E.Z.N.A. Water DNA Kit（OMEGA Bio-Tek Inc., USA），按照生产商的说明操作。提取的 DNA 在-80°C 冰箱中保存，以备后续分析。16S rRNA 扩增子通过引物对 515F/806R 扩增（515F: 5′-NNNNNNNNGTGTGCCAGCMGCCGCGGTAA-3′，806R: 5′-GGACTACHVGGGTWTCTAAT-3′），该引物针对 16S rRNA 基因的 V4 高变区[57]。16S rRNA 扩增子的高通量测序在北京诺禾致源生物信息科技有限公司（Novogene Bioinformatics Company）使用 Illumina MiSeq 平台完成。

Sequencing data analysis  测序数据分析

Paired-end reads were assigned based on the unique barcodes of samples, which were subsequently truncated by cutting off the barcode and primer sequence. The paired-end reads were merged using FLASH (V1.2.7) into raw tags. Quality filtering on the raw tags was performed to obtain high-quality clean tags according to QIIME (V1.7.0). The tags were compared with the reference database (Gold database) using a UCHIME algorithm to detect chimera sequences. The chimera sequences were removed to obtain the effective tags. Sequence analyses were performed using Uparse software (Uparse v7.0.1001). Sequences with ≥ 97% similarity were assigned to the same OTUs. The representative sequence for each OTU was screened for further annotation. For each representative sequence, the GreenGene Database was used based on an RDP classifier (Version 2.2) algorithm to annotate taxonomic information. Alpha diversity indices (Chao1 and ACE) were applied to analyze bacterial diversity. All these indices were calculated with QIIME (Version 1.7.0) and displayed with boxplots drawn by R software (Version 2.15.3).
根据样本的独特条形码对双端读长进行分配，随后通过截断条形码和引物序列进行裁剪。通过使用 FLASH (V1.2.7) 将双端读长拼接成原始标签（raw tags）。根据 QIIME (V1.7.0) 对原始标签进行质量过滤，获得高质量的清洁标签（clean tags）。利用 UCHIME 算法将标签与参考数据库（Gold 数据库）进行比对以检测嵌合序列，并去除嵌合序列以获得有效标签（effective tags）。序列分析使用 Uparse 软件（Uparse v7.0.1001）完成，相似度≥97%的序列被归为同一 OTU。每个 OTU 的代表性序列被筛选用于进一步注释。对于每个代表性序列，基于 RDP 分类器（Version 2.2）算法，使用 GreenGene 数据库进行分类信息注释。通过 Chao1 和 ACE 等 Alpha 多样性指数分析细菌多样性。所有指数均使用 QIIME (Version 1.7.0) 计算，并通过 R 软件 (Version 2.15.3) 绘制箱线图显示。

Data analysis  数据分析

With regard to the nutrients remove rate and the bacterial abundance, statistical significance was assessed by analysis of variance (ANOVA) followed by Fisher’s post hoc test using the IBM SPSS Statistics 21.0 program (IBM, Armonk, New York, USA); while, the statistical test used to compare the indices of microbial diversities was the Wilcoxon signed-rank test. A P value of less than 0.05 was considered as statistically significant.
关于营养物去除率和细菌丰度的统计显著性，通过方差分析（ANOVA）并结合 Fisher 事后检验进行评估，采用 IBM SPSS Statistics 21.0 程序（IBM，美国纽约阿蒙克）。用于比较微生物多样性指数的统计检验为 Wilcoxon 符号秩检验。P 值小于 0.05 被认为具有统计学显著性。