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Chapter 14  第 14 章

Scale-Up of Phytosterols Bioconversion into Androstenedione
大规模生物转化植物固醇为雄烯二酮

Sonia Martínez-Cámara, Manuel de la Torre, José-Luis Barredo, and Marta Rodríguez-Sáiz
索尼亚·马丁内斯-卡马拉，曼努埃尔·德拉·托雷，何塞-路易斯·巴雷多，马尔塔·罗德里格斯-塞兹

Abstract  摘要

Phytosterols, coming as a by-product of vegetable oils or wood pulp, contain the cyclopentanoperhydrophenanthrene nucleus and can be bioconverted into steroid intermediates by removing the C17 side chain. This chapter shows the scale-up, from flask to bioreactor, of phytosterols bioconversion into 4 -androstene-3,17-dione (androstenedione; AD) using Mycolicibacterium neoaurum B-3805. Due to the fact that phytosterols and AD are nearly insoluble in water, two-phase systems and the use of chemically modified cyclodextrins have been described as methods to solve it. Here, we use a water-oil two-phase system that allows the bioconversion of up to 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} of phytosterols into AD in 5 L and 20 L bioreactors.
植物固醇作为植物油或木浆的副产品，包含环戊烷多氢苯蒽核心结构，可通过去除 C17 侧链生物转化为类固醇中间体。本章展示了使用放线菌属新近菌株 B-3805，从烧瓶到生物反应器规模上将植物固醇生物转化为 4-雄烯-3,17-二酮（雄烯二酮；AD）的过程。由于植物固醇和雄烯二酮在水中几乎不溶，已有使用两相系统和化学改性环糊精的方法来解决这一问题。在此，我们使用水-油两相系统，可在 5 升和 20 升生物反应器中将高达 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} 的植物固醇生物转化为雄烯二酮。

Key words Bioconversion, Phytosterols, Fermentation, 4-Androstene-3,17-dione, Androstenedione, AD, Mycolicibacterium neoaurum, Mycobacterium neoaurum, Two-phase system
关键词 生物转化、植物甾醇、发酵、4-雄烯-3,17-二酮、雄烯二酮、AD、新鹿分枝杆菌、新鹿分枝杆菌、双相系统

1 Introduction  1 引言

Androst-4-ene-3,17-dione (androstenedione; AD) is a key intermediate in the synthesis of steroids such as testosterone, estradiol, progesterone, cortisone, prednisone, or prednisolone, which have great interest for the pharmaceutical companies [1-3]. In fact, the global market for steroids was significantly large in 2020, and it is expected to reach the value of US $ 17 $ 17 $17\$ 17 billion by the end of 2025 with an annual growth rate of around 3 % 3 % 3%3 \%. Key factors responsible for driving revenue growth are the increasing number of applications in medical issues and changes in the market dynamics due to the outbreak of the COVID-19 pandemic. In fact, dexamethasone treatment was probed to reduce mortality in patients with COVID19 who were receiving respiratory support.
雄烯-4-烯-3,17-二酮（雄烯二酮；AD）是合成睾酮、雌二醇、孕酮、可的松、泼尼松或泼尼龙等类固醇的关键中间体，对制药公司具有极大的兴趣[1-3]。事实上，2020 年类固醇的全球市场规模相当大，预计到 2025 年底将达到美元 $ 17 $ 17 $17\$ 17 十亿，年增长率约为 3 % 3 % 3%3 \% 。推动收入增长的关键因素是医疗应用增加以及由 COVID-19 疫情引起的市场动态变化。事实上，地塞米松治疗被证实可以降低接受呼吸支持的 COVID-19 患者的死亡率。
Anabolic steroids, including AD, androsta-1,4-diene-3,17dione (androstadienedione; ADD), and derivatives, are used to treat medical conditions such as late puberty, hormonal imbalance, breast cancer in woman, or muscle loss of patients suffering from AIDS and cancer. This type of steroids marks a faster growth rate in developed countries, especially in North America due to an increase in their questioned use as enhancer for athletic performance. According to MarketWatch, the global AD market was valued at USD 190 million in 2018 and is estimated to reach USD 210 million by 2025 [4]. The growing demand of the world market for these and other steroidal products has focused pharmaceutical research on producing high-value steroid precursors through the use of efficient eco-friendly technologies with low production costs, as an alternative to the more environmentally unfriendly chemical processes [5].
包括 AD、雄烯-1,4-二烯-3,17-二酮（雄烯二烯二酮；ADD）在内的同化类固醇，被用于治疗晚期青春期、激素失衡、女性乳腺癌或艾滋病和癌症患者的肌肉流失等医疗状况。这类类固醇在发达国家的增长速度更快，尤其是在北美地区，这主要是由于其被质疑用作运动员性能增强剂。据 MarketWatch 报道，2018 年全球 AD 市场价值为 1.9 亿美元，预计到 2025 年将达到 2.1 亿美元[4]。世界市场对这些和其他类固醇产品的不断增长的需求，促使制药研究专注于通过使用高效的环保技术和低成本生产工艺，生产高价值的类固醇前体，作为更加不环保的化学工艺的替代方案[5]。
The success of a fermentation-based process depends on the process cost (raw materials, utilities, manpower, indirect costs), the fermentation yield, and the market price. In this way, the use of phytosterols, a by-product of oil or wood pulp industry with similar characteristics to cholesterol such as structural behavior and functionality, as a starting material to produce steroid intermediates with Mycolicibacterium sp. in a single fermentation step, allows for the development of highly competitive processes in the global market [6-8].
发酵型工艺的成功取决于工艺成本（原材料、公用设施、人工、间接成本）、发酵收率以及市场价格。通过利用植物固醇这一石油或木浆工业的副产品，其具有与胆固醇相似的结构行为和功能特征，作为原料通过单一发酵步骤使用放线菌属细菌生产类固醇中间体，可以开发出在全球市场中具有高度竞争力的工艺流程[6-8]。
Mycolicibacterium sp., traditionally known as Mycobacterium sp., possesses the ability to catabolize naturally occurring 3- β β beta\beta-hydroxy- Δ 5 Δ 5 Delta5\Delta 5-sterols (such as cholesterol, β β beta\beta-sitosterol, etc.) or a mixture of sterols (phytosterols) to carbon dioxide and water, being able to accumulate a variety of steroidal intermediates during the degradation process [9-11]. New mutants have been obtained by classical mutagenesis or synthetic biology to block the catabolic pathway of phytosterol, favoring the accumulation of steroid intermediates of pharmaceutical value (Fig. 1) [12, 13]. Among them, Mycolicibacterium neoaurum NRRL-3805 (previously known as Mycobacterium neoaurum NRRL-3805), an AD producing strain obtained by mutagenesis from a soil isolate producing ADD, is found [14, 15]. This mutant transforms phytosterols into AD with a low content of ADD because of the partial failure of the dehydrogenation at Cl . The genome has been recently sequenced showing a circular chromosome of 5 , 421 , 338 5 , 421 , 338 5,421,3385,421,338 bp containing 4844 protein-coding gene [16].
传统上被称为放线菌属的放线菌 sp.具有分解天然存在的 3- β β beta\beta -羟基- Δ 5 Δ 5 Delta5\Delta 5 -甾醇（如胆固醇、 β β beta\beta -β-谷甾醇等）或甾醇混合物（植物甾醇）的能力，能够在降解过程中积累各种甾体中间产物[9-11]。通过传统诱变或合成生物学方法获得的新突变体能够阻断植物甾醇的分解途径，有利于积累具有药用价值的甾体中间产物（图 1）[12, 13]。其中，放线菌新繁殖株 NRRL-3805（之前称为放线菌新繁殖株 NRRL-3805），是一株通过诱变从土壤分离株获得的 AD 生产菌株，并被发现[14, 15]。该突变体将植物甾醇转化为 AD，且 ADD 含量较低，这是由于 C1 位点脱氢过程部分失败。该基因组最近已测序，呈现为一条 5 , 421 , 338 5 , 421 , 338 5,421,3385,421,338 bp 的环形染色体，包含 4844 个蛋白质编码基因[16]。
However, the industrial biotechnological processes for AD and other steroids are severely limited by the poor water solubility of phytosterols and the inhibition or toxicity that both, substrate and products, can cause to the microorganism [17]. Steroid-producing companies are focused on dealing with these obstacles, including
然而，对于 AD 和其他类固醇的工业生物技术工艺，受到植物固醇水溶性差以及底物和产物对微生物可能造成的抑制或毒性的严重限制[17]。类固醇生产公司正致力于解决这些障碍，包括
Fig. 1 Steroid bioconversions (blue arrows) using phytosterols as a starting material, and chemical processes (red arrows) for production of steroid Active Pharmaceutical Ingredients (APIs) from steroid intermediates
图 1 以植物固醇为起始原料的类固醇生物转化（蓝色箭头），以及从类固醇中间体生产类固醇活性药物成分（API）的化学工艺（红色箭头）
the use of biphasic systems, surfactants, liquid polymers, cyclodextrins, ionic liquids, organic solvents, resting cells, in situ product recovery, etc. to increase the phytosterol loading and achieve a more competitive process [4, 18-22].
使用双相系统、表面活性剂、液体聚合物、环糊精、离子液体、有机溶剂、静止细胞、原位产物回收等方法来提高植物固醇负载量并实现更具竞争力的工艺[4, 18-22]。
Carrying out a large-scale experimental fermentation is therefore costly, so pilot scale (from 5 to 40 L ) is a powerful tool before industrializing the laboratory results. A pilot plant mimics the industrial-scale fermentation process on a smaller scale, being cost-effective, environmentally aware, and the best option to explore potential changes in a production operation. Potential critical parameters of the AD bioconversion process, such as bioreactor design, raw materials, development of the vegetative phases, phytosterol loading, type of fermentation (batch, semi-batch, continuous, etc.), pH , oxygen, temperature, or viscosity, can be deeply studied at pilot scale before scaling up to industrial.
进行大规模实验发酵因此成本高昂，所以中试规模（从 5 到 40 升）是在工业化实验室结果之前的强大工具。中试装置可以在较小规模上模仿工业规模发酵过程，具有成本效益、环境意识，并且是探索生产运营潜在变化的最佳选择。发酵生物转化过程的潜在关键参数，如生物反应器设计、原材料、营养生长阶段、植物固醇负载量、发酵类型（批次、半批次、连续等）、pH 值、氧气、温度或粘度，可以在中试规模深入研究，然后再放大到工业规模。
Finally, the pilot-scale research allows the evaluation of the costs of different AD upstream and downstream technologies and to take a decision on process scale-up to comply with the quality commercial requirements.
最后，中试规模研究可以评估不同发酵上游和下游技术的成本，并就工艺放大做出决策，以满足商业质量要求。
In this chapter, we show a protocol for the bioconversion of phytosterols into AD at 5 L and 20 L scale, emphasizing those steps considered as critical points of the process.
在本章中，我们展示了从植物固醇到 AD 的生物转化协议，重点说明了过程中被视为关键点的步骤，并在 5 升和 20 升规模上进行。

2 Materials  2 材料

2.1 Vegetative Stages  2.1 营养生长阶段

2.1.1 First Vegetative and Second Vegetative Stages (Flask Scale)
2.1.1 第一营养生长阶段和第二营养生长阶段（烧瓶规模）
2.1.2 Third Vegetative Stage (5 L Bioreactor)
2.1.2 第三营养生长阶段（5 升生物反应器）

2.2 AD
Bioconversion at 5 L Scale
2.2 5 升规模的 AD 生物转化

2.3 AD  2.3 20 升规模的
Bioconversion at 20 L Scale
AD 生物转化
  1. Mycolicibacterium neoaurum B-3805 (CECT 3018; DSM 2967; NCIMB 11678).
    微茵菌属新霉菌株 B-3805（CECT 3018；DSM 2967；NCIMB 11678）。
  2. Erlenmeyer flasks: 500 mL for the first vegetative stage and 2 L for the second vegetative stage.
    500 mL 的锥形烧瓶用于第一个营养生长阶段，2 L 的锥形烧瓶用于第二个营养生长阶段。
  3. Growth medium: 54 g / L 54 g / L 54g//L54 \mathrm{~g} / \mathrm{L} of molasses, 5.4 g / L 5.4 g / L 5.4g//L5.4 \mathrm{~g} / \mathrm{L} of NaNO 3 NaNO 3 NaNO_(3)\mathrm{NaNO}_{3}, 0.6 g / L 0.6 g / L 0.6g//L0.6 \mathrm{~g} / \mathrm{L} of NH 4 H 2 PO 4 NH 4 H 2 PO 4 NH_(4)H_(2)PO_(4)\mathrm{NH}_{4} \mathrm{H}_{2} \mathrm{PO}_{4}, and 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} of soya oil. pH adjusted to 7.0.
    生长培养基： 54 g / L 54 g / L 54g//L54 \mathrm{~g} / \mathrm{L} 糖蜜， 5.4 g / L 5.4 g / L 5.4g//L5.4 \mathrm{~g} / \mathrm{L} 的 NaNO 3 NaNO 3 NaNO_(3)\mathrm{NaNO}_{3} ， 0.6 g / L 0.6 g / L 0.6g//L0.6 \mathrm{~g} / \mathrm{L} 的 NH 4 H 2 PO 4 NH 4 H 2 PO 4 NH_(4)H_(2)PO_(4)\mathrm{NH}_{4} \mathrm{H}_{2} \mathrm{PO}_{4} ，以及 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} 的大豆油。pH 调节至 7.0。
  4. Flow laminar cabinet.  层流柜
  5. Orbital shaker.  摇床
  6. Growth medium: 54 g / L 54 g / L 54g//L54 \mathrm{~g} / \mathrm{L} of molasses, 5.4 g / L 5.4 g / L 5.4g//L5.4 \mathrm{~g} / \mathrm{L} of NaNO 3 NaNO 3 NaNO_(3)\mathrm{NaNO}_{3}, 0.6 g / L 0.6 g / L 0.6g//L0.6 \mathrm{~g} / \mathrm{L} of NH 4 H 2 PO 4 NH 4 H 2 PO 4 NH_(4)H_(2)PO_(4)\mathrm{NH}_{4} \mathrm{H}_{2} \mathrm{PO}_{4}, and 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} of soya oil. pH adjusted to 7.0.
    生长培养基： 54 g / L 54 g / L 54g//L54 \mathrm{~g} / \mathrm{L} 糖蜜， 5.4 g / L 5.4 g / L 5.4g//L5.4 \mathrm{~g} / \mathrm{L} 的 NaNO 3 NaNO 3 NaNO_(3)\mathrm{NaNO}_{3} ， 0.6 g / L 0.6 g / L 0.6g//L0.6 \mathrm{~g} / \mathrm{L} 的 NH 4 H 2 PO 4 NH 4 H 2 PO 4 NH_(4)H_(2)PO_(4)\mathrm{NH}_{4} \mathrm{H}_{2} \mathrm{PO}_{4} ，以及 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} 的大豆油。pH 调节至 7.0。
  7. Fermentor Bioflo 3000, or equivalent, with a 5 L glass vessel (New Brunswick Scientific).
    Bioflo 3000 发酵罐，或同等设备，带有 5 L 玻璃容器（新不伦瑞克科学公司）。
  8. Inoculation flask: sterile Erlenmeyer 2 L flask with silicone tube and stainless-steel needle.
    接种烧瓶：带有硅胶管和不锈钢针的 2 升无菌的锥形瓶。
  9. Bioconversion medium: 2 g / L 2 g / L 2g//L2 \mathrm{~g} / \mathrm{L} of NH 4 NO 3 , l g / L NH 4 NO 3 , l g / L NH_(4)NO_(3),lg//L\mathrm{NH}_{4} \mathrm{NO}_{3}, \mathrm{l} \mathrm{g} / \mathrm{L} of KH 2 PO 4 , 2 g / L KH 2 PO 4 , 2 g / L KH_(2)PO_(4),2g//L\mathrm{KH}_{2} \mathrm{PO}_{4}, 2 \mathrm{~g} / \mathrm{L} of K 2 HPO 4 , 0.2 g / L K 2 HPO 4 , 0.2 g / L K_(2)HPO_(4),0.2g//L\mathrm{K}_{2} \mathrm{HPO}_{4}, 0.2 \mathrm{~g} / \mathrm{L} of KCl , 0.3 g / L KCl , 0.3 g / L KCl,0.3g//L\mathrm{KCl}, 0.3 \mathrm{~g} / \mathrm{L} of CaCl 2 , 150 g / L CaCl 2 , 150 g / L CaCl_(2),150g//L\mathrm{CaCl}_{2}, 150 \mathrm{~g} / \mathrm{L} of soya oil, and 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} of phytosterol from pine tree (Arboris). Add 1 mL / L 1 mL / L 1mL//L1 \mathrm{~mL} / \mathrm{L} from a micronutrient solution ( 11 g / L 11 g / L (11(g)//L:}\left(11 \mathrm{~g} / \mathrm{L}\right. of ZnSO 4 , 1 g / L ZnSO 4 , 1 g / L ZnSO_(4),1g//L\mathrm{ZnSO}_{4}, 1 \mathrm{~g} / \mathrm{L} of FeSO 4 , 6 g / L FeSO 4 , 6 g / L FeSO_(4),6g//L\mathrm{FeSO}_{4}, 6 \mathrm{~g} / \mathrm{L} of MnSO 4 MnSO 4 MnSO_(4)\mathrm{MnSO}_{4}, 0.04 g / L 0.04 g / L 0.04g//L0.04 \mathrm{~g} / \mathrm{L} of CuSO 4 , 0.3 g / L CuSO 4 , 0.3 g / L CuSO_(4),0.3g//L\mathrm{CuSO}_{4}, 0.3 \mathrm{~g} / \mathrm{L} of CoSO 4 , 0.03 g / L CoSO 4 , 0.03 g / L CoSO_(4),0.03g//L\mathrm{CoSO}_{4}, 0.03 \mathrm{~g} / \mathrm{L} of H 3 PO 4 H 3 PO 4 H_(3)PO_(4)\mathrm{H}_{3} \mathrm{PO}_{4}, and 0.001 g / L 0.001 g / L 0.001g//L0.001 \mathrm{~g} / \mathrm{L} of KI ). pH adjusted to 7.0 .
    生物转化培养基： 2 g / L 2 g / L 2g//L2 \mathrm{~g} / \mathrm{L} 的 NH 4 NO 3 , l g / L NH 4 NO 3 , l g / L NH_(4)NO_(3),lg//L\mathrm{NH}_{4} \mathrm{NO}_{3}, \mathrm{l} \mathrm{g} / \mathrm{L} 的 KH 2 PO 4 , 2 g / L KH 2 PO 4 , 2 g / L KH_(2)PO_(4),2g//L\mathrm{KH}_{2} \mathrm{PO}_{4}, 2 \mathrm{~g} / \mathrm{L} 的 K 2 HPO 4 , 0.2 g / L K 2 HPO 4 , 0.2 g / L K_(2)HPO_(4),0.2g//L\mathrm{K}_{2} \mathrm{HPO}_{4}, 0.2 \mathrm{~g} / \mathrm{L} 的 KCl , 0.3 g / L KCl , 0.3 g / L KCl,0.3g//L\mathrm{KCl}, 0.3 \mathrm{~g} / \mathrm{L} 的 CaCl 2 , 150 g / L CaCl 2 , 150 g / L CaCl_(2),150g//L\mathrm{CaCl}_{2}, 150 \mathrm{~g} / \mathrm{L} 的大豆油，以及来自松树（Arboris）的 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} 植物固醇。从微量营养液中添加 1 mL / L 1 mL / L 1mL//L1 \mathrm{~mL} / \mathrm{L} ，其中包括 ( 11 g / L 11 g / L (11(g)//L:}\left(11 \mathrm{~g} / \mathrm{L}\right. 的 ZnSO 4 , 1 g / L ZnSO 4 , 1 g / L ZnSO_(4),1g//L\mathrm{ZnSO}_{4}, 1 \mathrm{~g} / \mathrm{L} 的 FeSO 4 , 6 g / L FeSO 4 , 6 g / L FeSO_(4),6g//L\mathrm{FeSO}_{4}, 6 \mathrm{~g} / \mathrm{L} 的 MnSO 4 MnSO 4 MnSO_(4)\mathrm{MnSO}_{4} ， 0.04 g / L 0.04 g / L 0.04g//L0.04 \mathrm{~g} / \mathrm{L} 的 CuSO 4 , 0.3 g / L CuSO 4 , 0.3 g / L CuSO_(4),0.3g//L\mathrm{CuSO}_{4}, 0.3 \mathrm{~g} / \mathrm{L} 的 CoSO 4 , 0.03 g / L CoSO 4 , 0.03 g / L CoSO_(4),0.03g//L\mathrm{CoSO}_{4}, 0.03 \mathrm{~g} / \mathrm{L} 的 H 3 PO 4 H 3 PO 4 H_(3)PO_(4)\mathrm{H}_{3} \mathrm{PO}_{4} ，以及 0.001 g / L 0.001 g / L 0.001g//L0.001 \mathrm{~g} / \mathrm{L} 的碘化钾）。pH 调节至 7.0。
  10. Fermentor Bioflo 3000, or equivalent, with a 5 L glass vessel (New Brunswick Scientific).
    发酵罐 Bioflo 3000，或同等设备，配备 5 升玻璃容器（新不伦瑞克科学公司）。
  11. Blender.  搅拌器。
  12. Bioconversion medium: 2 g / L 2 g / L 2g//L2 \mathrm{~g} / \mathrm{L} of NH 4 NO 3 , l g / L NH 4 NO 3 , l g / L NH_(4)NO_(3),lg//L\mathrm{NH}_{4} \mathrm{NO}_{3}, \mathrm{l} \mathrm{g} / \mathrm{L} of KH 2 PO 4 , 2 g / L KH 2 PO 4 , 2 g / L KH_(2)PO_(4),2g//L\mathrm{KH}_{2} \mathrm{PO}_{4}, 2 \mathrm{~g} / \mathrm{L} of K 2 HPO 4 , 0.2 g / L K 2 HPO 4 , 0.2 g / L K_(2)HPO_(4),0.2g//L\mathrm{K}_{2} \mathrm{HPO}_{4}, 0.2 \mathrm{~g} / \mathrm{L} of KCl , 0.3 g / L KCl , 0.3 g / L KCl,0.3g//L\mathrm{KCl}, 0.3 \mathrm{~g} / \mathrm{L} of CaCl 2 , 150 g / L CaCl 2 , 150 g / L CaCl_(2),150g//L\mathrm{CaCl}_{2}, 150 \mathrm{~g} / \mathrm{L} of soya oil, and 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} of phytosterol from pine tree (Arboris). Add 1 mL / L 1 mL / L 1mL//L1 \mathrm{~mL} / \mathrm{L} from a micronutrient solution ( 11 g / L 11 g / L (11(g)//L:}\left(11 \mathrm{~g} / \mathrm{L}\right. of ZnSO 4 , 1 g / L ZnSO 4 , 1 g / L ZnSO_(4),1g//L\mathrm{ZnSO}_{4}, 1 \mathrm{~g} / \mathrm{L} of FeSO 4 , 6 g / L FeSO 4 , 6 g / L FeSO_(4),6g//L\mathrm{FeSO}_{4}, 6 \mathrm{~g} / \mathrm{L} of MnSO 4 MnSO 4 MnSO_(4)\mathrm{MnSO}_{4}, 0.04 g / L 0.04 g / L 0.04g//L0.04 \mathrm{~g} / \mathrm{L} of CuSO 4 , 0.3 g / L CuSO 4 , 0.3 g / L CuSO_(4),0.3g//L\mathrm{CuSO}_{4}, 0.3 \mathrm{~g} / \mathrm{L} of CoSO 4 , 0.03 g / L CoSO 4 , 0.03 g / L CoSO_(4),0.03g//L\mathrm{CoSO}_{4}, 0.03 \mathrm{~g} / \mathrm{L} of H 3 PO 4 H 3 PO 4 H_(3)PO_(4)\mathrm{H}_{3} \mathrm{PO}_{4}, and 0.001 g / L 0.001 g / L 0.001g//L0.001 \mathrm{~g} / \mathrm{L} of KI ). pH adjusted to 7.0 .
    生物转化培养基： 2 g / L 2 g / L 2g//L2 \mathrm{~g} / \mathrm{L} 的 NH 4 NO 3 , l g / L NH 4 NO 3 , l g / L NH_(4)NO_(3),lg//L\mathrm{NH}_{4} \mathrm{NO}_{3}, \mathrm{l} \mathrm{g} / \mathrm{L} 的 KH 2 PO 4 , 2 g / L KH 2 PO 4 , 2 g / L KH_(2)PO_(4),2g//L\mathrm{KH}_{2} \mathrm{PO}_{4}, 2 \mathrm{~g} / \mathrm{L} 的 K 2 HPO 4 , 0.2 g / L K 2 HPO 4 , 0.2 g / L K_(2)HPO_(4),0.2g//L\mathrm{K}_{2} \mathrm{HPO}_{4}, 0.2 \mathrm{~g} / \mathrm{L} 的 KCl , 0.3 g / L KCl , 0.3 g / L KCl,0.3g//L\mathrm{KCl}, 0.3 \mathrm{~g} / \mathrm{L} 的 CaCl 2 , 150 g / L CaCl 2 , 150 g / L CaCl_(2),150g//L\mathrm{CaCl}_{2}, 150 \mathrm{~g} / \mathrm{L} 的大豆油，以及来自松树（Arboris）的 20 g / L 20 g / L 20g//L20 \mathrm{~g} / \mathrm{L} 植物固醇。从微量营养液中添加 1 mL / L 1 mL / L 1mL//L1 \mathrm{~mL} / \mathrm{L} ，其中包括 ( 11 g / L 11 g / L (11(g)//L:}\left(11 \mathrm{~g} / \mathrm{L}\right. 的 ZnSO 4 , 1 g / L ZnSO 4 , 1 g / L ZnSO_(4),1g//L\mathrm{ZnSO}_{4}, 1 \mathrm{~g} / \mathrm{L} 的 FeSO 4 , 6 g / L FeSO 4 , 6 g / L FeSO_(4),6g//L\mathrm{FeSO}_{4}, 6 \mathrm{~g} / \mathrm{L} 的 MnSO 4 MnSO 4 MnSO_(4)\mathrm{MnSO}_{4} ， 0.04 g / L 0.04 g / L 0.04g//L0.04 \mathrm{~g} / \mathrm{L} 的 CuSO 4 , 0.3 g / L CuSO 4 , 0.3 g / L CuSO_(4),0.3g//L\mathrm{CuSO}_{4}, 0.3 \mathrm{~g} / \mathrm{L} 的 CoSO 4 , 0.03 g / L CoSO 4 , 0.03 g / L CoSO_(4),0.03g//L\mathrm{CoSO}_{4}, 0.03 \mathrm{~g} / \mathrm{L} 的 H 3 PO 4 H 3 PO 4 H_(3)PO_(4)\mathrm{H}_{3} \mathrm{PO}_{4} ，以及 0.001 g / L 0.001 g / L 0.001g//L0.001 \mathrm{~g} / \mathrm{L} 的碘化钾）。pH 调节至 7.0。
  13. Fermentor Biostat C , or equivalent, with a stainless-steel vessel of 20 L (about 15 L working volume) (Sartorius).
    发酵罐生物反应器 Biostat C，或同等设备，带有 20 L 的不锈钢容器（约 15 L 工作体积）（Sartorius）。
  14. Sterile tubing system for vegetative culture transfer.
    用于营养培养转移的无菌管路系统。

2.4 AD Analysis  2.4 AD 分析

2.4.1 AD Analysis by HPLC
2.4.1 通过高效液相色谱（HPLC）进行 AD 分析
2.4.2 AD Analysis by TLC
2.4.2 TLC 分析
  1. Androstenedione (4-androstene-3,17-dione, AD) 99.5% purity (Sigma-Aldrich).
    雄烯二酮（4-雄烯-3,17-二酮，AD）99.5%纯度（Sigma-Aldrich）。
  2. Butyl acetate.  丁酸乙酯。
  3. Acetonitrile HPLC grade.
    色谱级乙腈
  4. Analytical balance.  分析天平。
  5. HPLC equipment. Waters Alliance e2695 HPLC system with Waters 2998 Photodiode Array (PDA) Detector, or equivalent (Waters).
    高效液相色谱仪。Waters Alliance e2695 高效液相色谱系统，配备 Waters 2998 光电二极管阵列（PDA）检测器，或同等设备（Waters）。
  6. Column RP C18 Nucleosil 250 × 4.6 mM , 5 μ M 250 × 4.6 mM , 5 μ M 250 xx4.6mM,5muM250 \times 4.6 \mathrm{mM}, 5 \mu \mathrm{M} (with guard column), or equivalent.
    RP C18 Nucleosil 250 × 4.6 mM , 5 μ M 250 × 4.6 mM , 5 μ M 250 xx4.6mM,5muM250 \times 4.6 \mathrm{mM}, 5 \mu \mathrm{M} 色谱柱（带保护柱），或同等级色谱柱。
  7. Mobile phase: Acetonitrile/water ( 52 / 48 ) ( 52 / 48 ) (52//48)(52 / 48) with acetic acid 0.01 % 0.01 % 0.01%0.01 \%.
    流动相：乙腈/水 ( 52 / 48 ) ( 52 / 48 ) (52//48)(52 / 48) 并加入乙酸 0.01 % 0.01 % 0.01%0.01 \% 。
  8. Ethyl acetate.  乙酸乙酯。
  9. TLC chamber for 20 × 20 cm 20 × 20 cm 20 xx20cm20 \times 20 \mathrm{~cm} aluminum sheets.
    用于 20 × 20 cm 20 × 20 cm 20 xx20cm20 \times 20 \mathrm{~cm} 铝片的薄层色谱室。
  10. TLC Silica gel 60 F354 aluminum sheets 20 × 20 cm 20 × 20 cm 20 xx20cm20 \times 20 \mathrm{~cm} (Merck).
    TLC 硅胶 60 F354 铝片 20 × 20 cm 20 × 20 cm 20 xx20cm20 \times 20 \mathrm{~cm} （默克公司）。
  11. Mobile phase: Heptane/ethyl acetate ( 1 / 1 1 / 1 1//11 / 1 ).
    流动相：庚烷/乙酸乙酯（ 1 / 1 1 / 1 1//11 / 1 ）。
  12. Development solution: 20 % H 2 SO 4 20 % H 2 SO 4 20%H_(2)SO_(4)20 \% \mathrm{H}_{2} \mathrm{SO}_{4}.
    开发解决方案： 20 % H 2 SO 4 20 % H 2 SO 4 20%H_(2)SO_(4)20 \% \mathrm{H}_{2} \mathrm{SO}_{4} 。
  13. Heater plate.  加热板。

2.5 General
Equipment  2.5 常规设备

  1. Desktop centrifuge.  台式离心机。
  2. Swinging head centrifuge.
    摇摆头离心机。
  3. Microcentrifuge.  微量离心机。
  4. pH meter.  pH 计。
  5. Autoclave.  高压灭菌器。
  6. Microscope.  显微镜。
  7. Graduate tubes.  刻度试管。
  8. Magnetic stirrer.  磁力搅拌器。

3 Methods  3 种方法

3.1 Vegetative
Stages  3.1 营养生长阶段

The vegetative stages are necessary in any fermentation process to obtain enough biomass for the successful productive stage. The number of seed stages and incubation time depends on the growth rate of the microorganism and on the size of the bioreactor. In the case of AD bioconversion process, about 10 % ( v / v ) 10 % ( v / v ) 10%(v//v)10 \%(\mathrm{v} / \mathrm{v}) seeding is required to inoculate the productive stage. Therefore, a bioconversion process in a 5 L bioreactor with a working volume of 3 L will need 300 mL of vegetative stage.
营养生长阶段在任何发酵过程中都是必要的，目的是获得足够的生物质以成功进行生产阶段。种子阶段的数量和培养时间取决于微生物的生长速率和生物反应器的大小。在厌氧消化生物转化过程中，需要约 10 % ( v / v ) 10 % ( v / v ) 10%(v//v)10 \%(\mathrm{v} / \mathrm{v}) 接种来接种生产阶段。因此，在工作体积为 3 L 的 5 L 生物反应器中，生物转化过程将需要 300 mL 的营养生长阶段。
From a vial with 1 mL of frozen ( − 80 ∘ C ) − 80 ∘ C (-80^(@)C)\left(-80^{\circ} \mathrm{C}\right) suspension as initial seeding, two growth stages (first vegetative and second vegetative) are necessary to obtain enough biomass to inoculate the
从具有 1 mL 冷冻 ( − 80 ∘ C ) − 80 ∘ C (-80^(@)C)\left(-80^{\circ} \mathrm{C}\right) 悬浮液的小瓶作为初始接种，需要两个生长阶段（第一营养生长阶段和第二营养生长阶段）以获得足够的生物质来接种
fermentative stage. The process in a 20 L bioreactor with a working volume of 15 L includes an additional seed stage (third vegetative) to get 1.5 L .
发酵阶段。在一个工作体积为 15 L 的 20 L 生物反应器中，包括一个额外的种子阶段（第三营养阶段）以获得 1.5 L。
3.1.1 Mycolicibacterium neoaurum B-3805 First Vegetative Stage (100 mL)
3.1.1 迈考利氏菌新霉素 B-3805 第一营养阶段（100 mL）

1. 从培养皿上取一个迈考利氏菌新霉素 B-3805 的菌落，或取 1 mL 冷冻悬液（保存于 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} ），将其接种于 500 mL 烧瓶中的 100 mL 生长培养基中（参见注释 1 和 2）。 2. 在轨道振荡器中培养，行程 5 cm，温度 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} ，转速 250 rpm，培养 68-74 h（参见注释 3）。 3. 取样测定培养液 pH 值。72 h 培养后的预期值应约为 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2 。 4. 将 10 mL 培养液转移到刻度管中，在摇摆头离心机中以 4000 × g 4000 × g 4000 xx g4000 \times g 离心 10 分钟。 5. 通过将生物质含量表示为离心体积的百分比来计算沉淀体积（压实细胞体积；PCV）。转移到第二营养阶段的最佳 PCV 约为 5 − 6 % 5 − 6 % 5-6%5-6 \% 。
1. Take a colony of M. neoaurum B-3805 from a Petri plate or 1 mL of frozen suspension kept at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C}, and seed a 500 mL flask with 100 mL of growth medium (see Notes 1 and 2).
2. Incubate in an orbital shaker with 5 cm stroke at 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} and 250 rpm for 68-74 h (see Note 3).
3. Take a sample of the broth and measure the pH . The expected value after 72 h incubation should be about 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2.
4. Transfer 10 mL of the broth to a graduated tube and centrifuge the sample in a swinging head centrifuge at 4000 × g 4000 × g 4000 xx g4000 \times g for 10 min .
5. Calculate the pellet volume (Packed Cell Volume; PCV) by expressing the biomass content as a percentage of the centrifuged volume. The optimal PCV to transfer to the second vegetative stage is around 5 − 6 % 5 − 6 % 5-6%5-6 \%.
1. Take a colony of M. neoaurum B-3805 from a Petri plate or 1 mL of frozen suspension kept at -80^(@)C, and seed a 500 mL flask with 100 mL of growth medium (see Notes 1 and 2). 2. Incubate in an orbital shaker with 5 cm stroke at 31^(@)C and 250 rpm for 68-74 h (see Note 3). 3. Take a sample of the broth and measure the pH . The expected value after 72 h incubation should be about 6.8+-0.2. 4. Transfer 10 mL of the broth to a graduated tube and centrifuge the sample in a swinging head centrifuge at 4000 xx g for 10 min . 5. Calculate the pellet volume (Packed Cell Volume; PCV) by expressing the biomass content as a percentage of the centrifuged volume. The optimal PCV to transfer to the second vegetative stage is around 5-6%.| 1. Take a colony of M. neoaurum B-3805 from a Petri plate or 1 mL of frozen suspension kept at $-80^{\circ} \mathrm{C}$, and seed a 500 mL flask with 100 mL of growth medium (see Notes 1 and 2). | | :--- | | 2. Incubate in an orbital shaker with 5 cm stroke at $31^{\circ} \mathrm{C}$ and 250 rpm for 68-74 h (see Note 3). | | 3. Take a sample of the broth and measure the pH . The expected value after 72 h incubation should be about $6.8 \pm 0.2$. | | 4. Transfer 10 mL of the broth to a graduated tube and centrifuge the sample in a swinging head centrifuge at $4000 \times g$ for 10 min . | | 5. Calculate the pellet volume (Packed Cell Volume; PCV) by expressing the biomass content as a percentage of the centrifuged volume. The optimal PCV to transfer to the second vegetative stage is around $5-6 \%$. |
3.1.2 M. neoaurum B B BB 3805 Second Vegetative Stage ( 300 mL )
3.1.2 迈考利氏菌新霉素 B B BB 3805 第二营养阶段（300 mL）

1. 取 30 mL 第一阶段营养培养液，接种到装有 300 mL 生长培养基的 2 L 烧瓶中（参见注释 2）。2. 在行程为 5 cm 的摇床上，以 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} 和 200 rpm 孵育 24-30 小时（参见注释 4）。3. 取培养液样本，按照 3.1.1 小节测量 pH 和 PCV。24 小时孵育后的预期 pH 值约为 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2 和 PCV > 5 % PCV > 5 % PCV > 5%\mathrm{PCV}>5 \% 。4. 将全部培养液转移到第三阶段营养培养或生物转化阶段（5 L 生物反应器）。
1. Take 30 mL of the first vegetative culture and seed a 2 L flask with 300 mL of growth medium (see Note 2).
2. Incubate in an orbital shaker of 5 cm stroke at 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} and 200 rpm for 24-30 h (see Note 4).
3. Take a sample of the broth and measure pH and PCV according to the Subheading 3.1.1. The expected pH after 24 h incubation is about 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2 and PCV > 5 % PCV > 5 % PCV > 5%\mathrm{PCV}>5 \%.
4. Transfer the total culture broth to the third vegetative stage or to the bioconversion stage ( 5 L bioreactor).
1. Take 30 mL of the first vegetative culture and seed a 2 L flask with 300 mL of growth medium (see Note 2). 2. Incubate in an orbital shaker of 5 cm stroke at 31^(@)C and 200 rpm for 24-30 h (see Note 4). 3. Take a sample of the broth and measure pH and PCV according to the Subheading 3.1.1. The expected pH after 24 h incubation is about 6.8+-0.2 and PCV > 5%. 4. Transfer the total culture broth to the third vegetative stage or to the bioconversion stage ( 5 L bioreactor).| 1. Take 30 mL of the first vegetative culture and seed a 2 L flask with 300 mL of growth medium (see Note 2). | | :--- | | 2. Incubate in an orbital shaker of 5 cm stroke at $31^{\circ} \mathrm{C}$ and 200 rpm for 24-30 h (see Note 4). | | 3. Take a sample of the broth and measure pH and PCV according to the Subheading 3.1.1. The expected pH after 24 h incubation is about $6.8 \pm 0.2$ and $\mathrm{PCV}>5 \%$. | | 4. Transfer the total culture broth to the third vegetative stage or to the bioconversion stage ( 5 L bioreactor). |
3.1.3 M. neoaurum B-3805 Third Vegetative Stage (5L)
3.1.3 M. neoaurum B-3805 第三阶段营养培养（5 L）		 The bioconversion process in a 20 L bioreactor needs a third vegetative stage to obtain enough biomass to inoculate the productive stage. This culture can be performed in a bioreactor instead of using several 2 L flasks, thus allowing to increase the parameter controls and to reduce the contamination risk during the transfer of the cultures from several flasks to the bioreactor.
20 L 生物反应器中的生物转化过程需要第三阶段营养培养以获得足够的生物量来接种生产阶段。相比使用多个 2 L 烧瓶，这种培养可以在生物反应器中进行，从而能够更好地控制参数并降低从多个烧瓶转移培养物到生物反应器时的污染风险。
Media and Bioreactor Preparation
培养基和生物反应器准备

1. 称量生长培养基的原材料，并依次溶解在温水中（最终体积 3 L）。2. 使用 20 % NaOH 20 % NaOH 20%NaOH20 \% \mathrm{NaOH} 调节 pH 至 7.0。3. 将培养基转移到生物反应器容器中，组装所有生物反应器部件，并在 121 ∘ C 121 ∘ C 121^(@)C121{ }^{\circ} \mathrm{C} 下对带浸入探头的样品进行 30 分钟高压灭菌。4. 灭菌后，将生物反应器放置在其控制台上，连接所有附件，并设置以下发酵参数：温度 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} ，空气流速 2.4 L / min 2.4 L / min 2.4L//min2.4 \mathrm{~L} / \mathrm{min} ，搅拌速度 450 rpm。
1. Weigh the raw materials of the growth medium and dissolve them sequentially in warm water (final volume 3 L ).
2. Adjust pH to 7.0 using 20 % NaOH 20 % NaOH 20%NaOH20 \% \mathrm{NaOH}.
3. Transfer the medium to the bioreactor vessel, assemble all bioreactor parts, and autoclave at 121 ∘ C 121 ∘ C 121^(@)C121{ }^{\circ} \mathrm{C} for 30 min with submerged probe.
4. After sterilization, place the bioreactor on its control console, connect all accessories, and set the next fermentation parameters: temperature 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C}, air flow rate 2.4 L / min 2.4 L / min 2.4L//min2.4 \mathrm{~L} / \mathrm{min}, and agitation 450 rpm .
1. Weigh the raw materials of the growth medium and dissolve them sequentially in warm water (final volume 3 L ). 2. Adjust pH to 7.0 using 20%NaOH. 3. Transfer the medium to the bioreactor vessel, assemble all bioreactor parts, and autoclave at 121^(@)C for 30 min with submerged probe. 4. After sterilization, place the bioreactor on its control console, connect all accessories, and set the next fermentation parameters: temperature 31^(@)C, air flow rate 2.4L//min, and agitation 450 rpm .| 1. Weigh the raw materials of the growth medium and dissolve them sequentially in warm water (final volume 3 L ). | | :--- | | 2. Adjust pH to 7.0 using $20 \% \mathrm{NaOH}$. | | 3. Transfer the medium to the bioreactor vessel, assemble all bioreactor parts, and autoclave at $121{ }^{\circ} \mathrm{C}$ for 30 min with submerged probe. | | 4. After sterilization, place the bioreactor on its control console, connect all accessories, and set the next fermentation parameters: temperature $31^{\circ} \mathrm{C}$, air flow rate $2.4 \mathrm{~L} / \mathrm{min}$, and agitation 450 rpm . |
fermentative stage. The process in a 20 L bioreactor with a working volume of 15 L includes an additional seed stage (third vegetative) to get 1.5 L . 3.1.1 Mycolicibacterium neoaurum B-3805 First Vegetative Stage (100 mL) "1. Take a colony of M. neoaurum B-3805 from a Petri plate or 1 mL of frozen suspension kept at -80^(@)C, and seed a 500 mL flask with 100 mL of growth medium (see Notes 1 and 2). 2. Incubate in an orbital shaker with 5 cm stroke at 31^(@)C and 250 rpm for 68-74 h (see Note 3). 3. Take a sample of the broth and measure the pH . The expected value after 72 h incubation should be about 6.8+-0.2. 4. Transfer 10 mL of the broth to a graduated tube and centrifuge the sample in a swinging head centrifuge at 4000 xx g for 10 min . 5. Calculate the pellet volume (Packed Cell Volume; PCV) by expressing the biomass content as a percentage of the centrifuged volume. The optimal PCV to transfer to the second vegetative stage is around 5-6%." 3.1.2 M. neoaurum B 3805 Second Vegetative Stage ( 300 mL ) "1. Take 30 mL of the first vegetative culture and seed a 2 L flask with 300 mL of growth medium (see Note 2). 2. Incubate in an orbital shaker of 5 cm stroke at 31^(@)C and 200 rpm for 24-30 h (see Note 4). 3. Take a sample of the broth and measure pH and PCV according to the Subheading 3.1.1. The expected pH after 24 h incubation is about 6.8+-0.2 and PCV > 5%. 4. Transfer the total culture broth to the third vegetative stage or to the bioconversion stage ( 5 L bioreactor)." 3.1.3 M. neoaurum B-3805 Third Vegetative Stage (5L) The bioconversion process in a 20 L bioreactor needs a third vegetative stage to obtain enough biomass to inoculate the productive stage. This culture can be performed in a bioreactor instead of using several 2 L flasks, thus allowing to increase the parameter controls and to reduce the contamination risk during the transfer of the cultures from several flasks to the bioreactor. Media and Bioreactor Preparation "1. Weigh the raw materials of the growth medium and dissolve them sequentially in warm water (final volume 3 L ). 2. Adjust pH to 7.0 using 20%NaOH. 3. Transfer the medium to the bioreactor vessel, assemble all bioreactor parts, and autoclave at 121^(@)C for 30 min with submerged probe. 4. After sterilization, place the bioreactor on its control console, connect all accessories, and set the next fermentation parameters: temperature 31^(@)C, air flow rate 2.4L//min, and agitation 450 rpm ."| | fermentative stage. The process in a 20 L bioreactor with a working volume of 15 L includes an additional seed stage (third vegetative) to get 1.5 L . | | :--- | :--- | | 3.1.1 Mycolicibacterium neoaurum B-3805 First Vegetative Stage (100 mL) | 1. Take a colony of M. neoaurum B-3805 from a Petri plate or 1 mL of frozen suspension kept at $-80^{\circ} \mathrm{C}$, and seed a 500 mL flask with 100 mL of growth medium (see Notes 1 and 2). <br> 2. Incubate in an orbital shaker with 5 cm stroke at $31^{\circ} \mathrm{C}$ and 250 rpm for 68-74 h (see Note 3). <br> 3. Take a sample of the broth and measure the pH . The expected value after 72 h incubation should be about $6.8 \pm 0.2$. <br> 4. Transfer 10 mL of the broth to a graduated tube and centrifuge the sample in a swinging head centrifuge at $4000 \times g$ for 10 min . <br> 5. Calculate the pellet volume (Packed Cell Volume; PCV) by expressing the biomass content as a percentage of the centrifuged volume. The optimal PCV to transfer to the second vegetative stage is around $5-6 \%$. | | 3.1.2 M. neoaurum $B$ 3805 Second Vegetative Stage ( 300 mL ) | 1. Take 30 mL of the first vegetative culture and seed a 2 L flask with 300 mL of growth medium (see Note 2). <br> 2. Incubate in an orbital shaker of 5 cm stroke at $31^{\circ} \mathrm{C}$ and 200 rpm for 24-30 h (see Note 4). <br> 3. Take a sample of the broth and measure pH and PCV according to the Subheading 3.1.1. The expected pH after 24 h incubation is about $6.8 \pm 0.2$ and $\mathrm{PCV}>5 \%$. <br> 4. Transfer the total culture broth to the third vegetative stage or to the bioconversion stage ( 5 L bioreactor). | | 3.1.3 M. neoaurum B-3805 Third Vegetative Stage (5L) | The bioconversion process in a 20 L bioreactor needs a third vegetative stage to obtain enough biomass to inoculate the productive stage. This culture can be performed in a bioreactor instead of using several 2 L flasks, thus allowing to increase the parameter controls and to reduce the contamination risk during the transfer of the cultures from several flasks to the bioreactor. | | Media and Bioreactor Preparation | 1. Weigh the raw materials of the growth medium and dissolve them sequentially in warm water (final volume 3 L ). <br> 2. Adjust pH to 7.0 using $20 \% \mathrm{NaOH}$. <br> 3. Transfer the medium to the bioreactor vessel, assemble all bioreactor parts, and autoclave at $121{ }^{\circ} \mathrm{C}$ for 30 min with submerged probe. <br> 4. After sterilization, place the bioreactor on its control console, connect all accessories, and set the next fermentation parameters: temperature $31^{\circ} \mathrm{C}$, air flow rate $2.4 \mathrm{~L} / \mathrm{min}$, and agitation 450 rpm . |
3.2 AD
Bioconversion at 5 L Scale
5 L 规模生物转化
3.2.1 Media and Bioreactor Preparation
3.2.1 培养基和生物反应器准备
3.2.2 Bioconversion of Phytosterol to AD
3.2.2 植物固醇生物转化为 AD
5. Calibrate the DO 2 DO 2 DO_(2)\mathrm{DO}_{2} probe assuming a theoretical rate of 100 % 100 % 100%100 \% before inoculating (maximal oxygen supply at the initial aeration, pressure, and agitation). A rate of 0 % 0 % 0%0 \% is obtained by briefly disconnecting the lead cable from the DO electrode and set to zero.
5. 在接种前，假设理论速率为 DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 校准探头（初始曝气、压力和搅拌时的最大氧气供应）。通过短暂断开溶解氧电极的引线电缆并设置为零，可获得 0 % 0 % 0%0 \% 的速率。
  1. Transfer 300 mL of a well-grown M. neoaurum B-3805 inoculum obtained according to Subheading 3.1.2 to a sterile inoculation flask in sterile conditions (see Note 2)
    在无菌条件下（参见注 2），将根据 3.1.2 小节获得的生长良好的 M. neoaurum B-3805 接种物 300 mL 转移到无菌接种烧瓶中。
  2. Inoculate the 5 L bioreactor by inserting the needle of the inoculation flask through the septum previously sterilized with a sporicidal solution.
    通过将接种烈瓶的针头插入之前已用杀孢子溶液消毒的隔膜，接种 5 L 生物反应器。
  3. Keep the incubation parameters defined in this Subheading and start the dissolved oxygen control over 30% (see Note 5).
    保持本小标题中定义的培养参数，并开始溶解氧控制在 30%以上（参见注释 5）。
  4. Take samples every 24 h in order to monitor M. neoaurum growth according to the procedure described in Subheading 3.1.1.
    每 24 小时取样一次，以根据 3.1.1 小节中描述的程序监测 M. neoaurum 的生长。
  5. The culture is ready to inoculate the bioconversion stage when pH is about 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2 and PCV > 5 % PCV > 5 % PCV > 5%\mathrm{PCV}>5 \%.
    当 pH 值约为 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2 且 PCV > 5 % PCV > 5 % PCV > 5%\mathrm{PCV}>5 \% 时，培养物即可接种生物转化阶段。
  6. Weigh the raw materials of the bioconversion medium, except phytosterol and oil, and dissolve them sequentially in warm water (see Note 6).
    称量生物转化培养基的原料，不包括植物固醇和油，并依次在温水中溶解（参见注释 6）。
  7. Adjust pH to 7.0 with 20 % NaOH 20 % NaOH 20%NaOH20 \% \mathrm{NaOH}.
    用 20 % NaOH 20 % NaOH 20%NaOH20 \% \mathrm{NaOH} 将 pH 调节至 7.0。
  8. Weigh oil and phytosterol separately, add them to the vessel of the bioreactor, and mix (see Note 7).
    分别称量油和植物甾醇，加入生物反应器容器中并混合（参见注释 7）。
  9. Add the bioconversion medium previously prepared to the bioreactor vessel. The final culture medium should be 3 L .
    向生物反应器容器中加入之前准备的生物转化培养基。最终培养基体积应为 3 L。
  10. Assemble all bioreactor parts and autoclave at 121 ∘ C 121 ∘ C 121^(@)C121{ }^{\circ} \mathrm{C} for 30 min with submerged probe.
    组装生物反应器所有部件，并将带浸入式探头的装置在 121 ∘ C 121 ∘ C 121^(@)C121{ }^{\circ} \mathrm{C} 下高压灭菌 30 分钟。
  11. After sterilization, place the bioreactor in its control console (see Note 8). Connect all accessories and set the following fermentation parameters: temperature 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C}, air flow rate 2.4 L / min 2.4 L / min 2.4L//min2.4 \mathrm{~L} / \mathrm{min}, and agitation 800 rpm .
    完成灭菌后，将生物反应器放置在其控制台中（参见注释 8）。连接所有配件，并设置以下发酵参数：温度 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} 、空气流速 2.4 L / min 2.4 L / min 2.4L//min2.4 \mathrm{~L} / \mathrm{min} ，以及搅拌速率 800 转/分。
  12. Calibrate the DO 2 DO 2 DO_(2)\mathrm{DO}_{2} probe according to Subheading 3.1.3.
    按照小节 3.1.3 的要求校准 DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 探针。
  13. Inoculate the 5 L bioreactor with 300 mL of a well-grown M. neodurum B-3805 inoculum (optimal pH and PCV parameters are 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2 and > 5 % > 5 % > 5%>5 \%, respectively) according to the procedure described in the Subheading 3.1.2.
    根据小节 3.1.2 中描述的程序，用 300 mL 生长良好的 M. neodurum B-3805 接种物接种 5 L 生物反应器（最佳 pH 和 PCV 参数分别为 6.8 ± 0.2 6.8 ± 0.2 6.8+-0.26.8 \pm 0.2 和 > 5 % > 5 % > 5%>5 \% ）。
    3.3 AD
Bioconversion at 20 L Scale
20 升规模的生物转化

3.3.1 Media and Bioreactor Preparation
3.3.1 培养基和生物反应器准备

3.3.2 Bioconversion of Phytosterol to AD
3.3.2 植物固醇转化为 AD
2. Incubate according to the following fermentation parameters: temperature 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C}, air flow rate 2.4 L / min 2.4 L / min 2.4L//min2.4 \mathrm{~L} / \mathrm{min}, agitation 800 rpm , and dissolved oxygen over 30 % 30 % 30%30 \% (see Note 9).
2. 根据以下发酵参数进行孵育：温度 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} ，空气流速 2.4 L / min 2.4 L / min 2.4L//min2.4 \mathrm{~L} / \mathrm{min} ，搅拌速度 800 rpm，溶解氧超过 30 % 30 % 30%30 \% （参见注释 9）。
3. Take samples every 8 h to check the absence of microbial contamination (see Note 2), pH (see Note 10), growth rate measured as PCV (Subheading 3.1.1) (see Note 11), phytosterol by TLC, and AD by TLC and HPLC (Subheading 3.4).
3. 每 8 小时采集样本，检查是否存在微生物污染（参见注释 2）、pH 值（参见注释 10）、生长速率（以 PCV 测量）（子标题 3.1.1）（参见注释 11）、薄层色谱法（TLC）检测植物固醇，以及薄层色谱法（TLC）和高效液相色谱法（HPLC）检测脂肪降解（子标题 3.4）。
4. Keep the bioconversion process until the residual concentration of phytosterol in the broth is under 1 g / L 1 g / L 1g//L1 \mathrm{~g} / \mathrm{L} (analyzed by TLC). The bioconversion should be completed in about 120 h (see Note 12).
4. 持续进行生物转化过程，直到发酵液中植物固醇的残留浓度低于 1 g / L 1 g / L 1g//L1 \mathrm{~g} / \mathrm{L} （通过薄层色谱法分析）。生物转化应在约 120 小时内完成（参见注释 12）。
  1. Weigh the raw materials of the bioconversion medium, except phytosterol and oil, and dissolve them sequentially by stirring in 4-5 L of warm water. Adjust pH to 7.0 with 20 % NaOH 20 % NaOH 20%NaOH20 \% \mathrm{NaOH}.
    称量生物转化培养基的原料，不包括植物固醇和油，然后在 4-5 升温水中依次搅拌溶解。用 20 % NaOH 20 % NaOH 20%NaOH20 \% \mathrm{NaOH} 调节 pH 值至 7.0。
  2. Place the pH and pO 2 probes in the corresponding inputs of the fermentation vessel and calibrate the pH probe with the appropriate buffer standard solutions ( 7.01 and 4.01 buffers).
    将 pH 探针和 pO2 探针放置在发酵罐的相应输入端，并使用适当的缓冲标准溶液（7.01 和 4.01 缓冲液）校准 pH 探针。
  3. Assembly the air filter and check that the position of the air entry is set to “Sterilization” position.
    组装空气过滤器，并确认空气入口位置设置为"灭菌"位置。
  4. Place the dissolved culture medium inside the bioreactor vessel, and check the pH with the bioreactor probe.
    将溶解的培养基置于生物反应器罐中，并使用生物反应器探头检查 pH 值。
  5. Adjust the volume to 12.5 L with distilled water and start stirring at 200 rpm .
    用蒸馏水将体积调节至 12.5 L，并开始以 200 rpm 的速度搅拌。
  6. Add the oil and phytosterol. The final volume should be approximately 15 L ( see Note 13).
    添加油和植物固醇。最终体积应约为 15 L（参见注释 13）。
  7. Close all the ports of the bioreactor vessel and perform a pressure test to check that the vessel is properly sealed (see Note 14). After that, open the air output to release pressure.
    关闭生物反应器罐的所有端口，并进行压力测试以检查罐是否密封良好（参见注释 14）。之后，打开空气输出口释放压力。
  8. Start the sterilization cycle according to the following sterilization parameters: temperature 121 ∘ C 121 ∘ C 121^(@)C121^{\circ} \mathrm{C}, agitation 200 rpm , time 30 min , and final temperature 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} (see Note 15).
    根据以下灭菌参数开始灭菌循环：温度 121 ∘ C 121 ∘ C 121^(@)C121^{\circ} \mathrm{C} ，搅拌 200 rpm，时间 30 分钟，最终温度 31 ∘ C 31 ∘ C 31^(@)C31^{\circ} \mathrm{C} （参见注释 15）。
  9. Once the sterilization process is finished, set the following fermentation parameters: temperature 31 ∘ C 31 ∘ C 31^(@)C31{ }^{\circ} \mathrm{C}, agitation 450 rpm , air flow rate 15 L / min 15 L / min 15L//min15 \mathrm{~L} / \mathrm{min}, and back pressure 0.8 bar.
    灭菌过程完成后，设置以下发酵参数：温度 31 ∘ C 31 ∘ C 31^(@)C31{ }^{\circ} \mathrm{C} ，搅拌 450 rpm，气流速率 15 L / min 15 L / min 15L//min15 \mathrm{~L} / \mathrm{min} ，背压 0.8 bar。
  10. Calibrate the DO 2 DO 2 DO_(2)\mathrm{DO}_{2} probe according to Subheading 3.1.3.
    根据小节 3.1.3 校准 DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 探针。
  11. Inoculate the bioconversion medium with 1.5 L of M. neoaurum B-3805 third vegetative (Subheading 3.1.3).
    用 1.5 L 的 M. neoaurum B-3805 第三代营养液接种生物转化培养基（小节 3.1.3）。
  12. Incubate the following fermentation parameters defined in Subheading 3.3.1 and start the dissolved oxygen control over 30% (see Note 16).
    按照子标题 3.3.1 中定义的发酵参数进行培养，并开始溶解氧控制在 30%以上（参见注释 16）。

3.4 Quantification of Sterols and AD
3.4 固醇和 AD 的定量

3.4.1 Analysis by HPLC
3.4.1 高效液相色谱（HPLC）分析
Sample Extraction  样品提取
Standard Preparation  标准制备
Parameter Analysis  参数分析
3.4.2 Analysis by TLC
3.4.2 薄层色谱分析
Sample Extraction  样品提取
3. Take samples every 8 h to check the absence of microbial contamination (see Note 2), pH (see Note 10), growth rate measured as PCV (Subheading 3.1.1) (see Note 11), phytosterol by TLC, and AD by TLC and HPLC (Subheading 3.4) ( see Note 17).
3. 每 8 小时取样检查是否存在微生物污染（参见注释 2）、pH 值（参见注释 10）、以 PCV 测量的生长速率（子标题 3.1.1）（参见注释 11）、薄层色谱法检测植物甾醇，以及薄层色谱法和高效液相色谱法检测 AD（子标题 3.4）（参见注释 17）。
4. Keep the bioconversion process until the residual concentration of phytosterol in the broth is under 1 g / L 1 g / L 1g//L1 \mathrm{~g} / \mathrm{L} (analyzed by TLC). The bioconversion should be completed in about 96 − 100 h 96 − 100 h 96-100h96-100 \mathrm{~h}.
4. 继续生物转化过程，直到发酵液中植物甾醇的剩余浓度低于 1 g / L 1 g / L 1g//L1 \mathrm{~g} / \mathrm{L} （通过薄层色谱法分析）。生物转化应在大约 96 − 100 h 96 − 100 h 96-100h96-100 \mathrm{~h} 内完成。
5. Harvest the bioconversion broth and begin the downstream process to purify AD.
5. 收获生物转化发酵液，开始后续工艺以纯化 AD。
  1. Take 20 mL of the bioconversion broth and mix with a magnetic stirrer for 10 min .
    取 20 mL 生物转化发酵液，并用磁力搅拌器搅拌 10 分钟。
  2. Take 100 μ L 100 μ L 100 muL100 \mu \mathrm{~L} of the stirred sample and mix with 900 μ L 900 μ L 900 muL900 \mu \mathrm{~L} of butyl acetate in a 2.2 mL microtube.
    取 100 μ L 100 μ L 100 muL100 \mu \mathrm{~L} 的搅拌样品，与 900 μ L 900 μ L 900 muL900 \mu \mathrm{~L} 的乙酸丁酯混合，放入 2.2 mL 微量离心管中。
  3. Stir at 50 ∘ C 50 ∘ C 50^(@)C50^{\circ} \mathrm{C} for 20 min .
    以 50 ∘ C 50 ∘ C 50^(@)C50^{\circ} \mathrm{C} 搅拌 20 分钟。
  4. Centrifuge at 10 , 000 × g 10 , 000 × g 10,000 xx g10,000 \times g for 10 min .
    以 10 , 000 × g 10 , 000 × g 10,000 xx g10,000 \times g 离心 10 分钟。
  5. Take a sample of 200 μ L 200 μ L 200 muL200 \mu \mathrm{~L} of the supernatant and prepare a second dilution in 800 μ L 800 μ L 800 muL800 \mu \mathrm{~L} of acetonitrile HPLC grade. The final dilution of the broth sample is 1 / 50 1 / 50 1//501 / 50.
    取 200 μ L 200 μ L 200 muL200 \mu \mathrm{~L} 的上清液，并在 800 μ L 800 μ L 800 muL800 \mu \mathrm{~L} 的色谱级乙腈中配制第二次稀释。发酵样品的最终稀释度为 1 / 50 1 / 50 1//501 / 50 。
  6. Weigh 10 mg of AD standard in a 10 mL volumetric flask and bring up to 70 % 70 % 70%70 \% of the final volume with acetonitrile HPLC grade.
    取 10 mg 的 AD 标准品置于 10 mL 容量瓶中，用色谱纯乙腈定容至 70 % 70 % 70%70 \% 体积。
  7. Sonicate until complete dissolution and bring up to 10 mL to achieve a concentration of 1 mg / mL 1 mg / mL 1mg//mL1 \mathrm{mg} / \mathrm{mL}.
    超声至完全溶解，并定容至 10 mL，以达到 1 mg / mL 1 mg / mL 1mg//mL1 \mathrm{mg} / \mathrm{mL} 浓度。
  8. Dilute 1 mL in a 50 mL volumetric flask with acetonitrile HPLC grade to achieve a final concentration of 20 mg / L 20 mg / L 20mg//L20 \mathrm{mg} / \mathrm{L}.
    取 1 mL 样品置于 50 mL 容量瓶中，用色谱纯乙腈稀释至最终浓度为 20 mg / L 20 mg / L 20mg//L20 \mathrm{mg} / \mathrm{L} 。
  9. Analyze 10 μ L 10 μ L 10 muL10 \mu \mathrm{~L} of the sample according to the following HPLC conditions: temperature 50 ∘ C 50 ∘ C 50^(@)C50^{\circ} \mathrm{C}, flow 1 mL / min 1 mL / min 1mL//min1 \mathrm{~mL} / \mathrm{min}, and wavelength 240 nm .
    按照以下 HPLC 条件分析 10 μ L 10 μ L 10 muL10 \mu \mathrm{~L} 样品：温度 50 ∘ C 50 ∘ C 50^(@)C50^{\circ} \mathrm{C} ，流速 1 mL / min 1 mL / min 1mL//min1 \mathrm{~mL} / \mathrm{min} ，波长 240 nm。
  10. Check the products according to their retention time (RT): ADD 5.4 min , testosterone 6.5 min , and AD 6.7 min (see Note 18).
    根据保留时间(RT)检查产物：ADD 5.4 分钟，睾酮 6.5 分钟，以及 AD 6.7 分钟（参见注释 18）。
  11. Take 20 mL of the bioconversion broth and mix with a magnetic stirrer for 10 min .
    取 20 mL 生物转化液，用磁力搅拌器搅拌 10 分钟。
  12. Take 100 μ L 100 μ L 100 muL100 \mu \mathrm{~L} of the stirred sample and mix with 900 μ L 900 μ L 900 muL900 \mu \mathrm{~L} of ethyl acetate in a 2.2 mL microtube (see Note 19).
    取 100 μ L 100 μ L 100 muL100 \mu \mathrm{~L} 的搅拌样品，与 900 μ L 900 μ L 900 muL900 \mu \mathrm{~L} 的乙酸乙酯混合，置于 2.2 mL 微量离心管中（参见注释 19）。
  13. Stir at 50 ∘ C 50 ∘ C 50^(@)C50^{\circ} \mathrm{C} for 20 min .
    在 50 ∘ C 50 ∘ C 50^(@)C50^{\circ} \mathrm{C} 下搅拌 20 分钟。
  14. Centrifuge at 10 , 000 × g 10 , 000 × g 10,000 xx g10,000 \times g for 10 min .
    以 10 分钟的速度在离心机中离心。
  15. Transfer the supernatant to another microtube and discard the pellet.
    将上清液转移到另一个微管中，丢弃沉淀。
TLC Development  薄层色谱（TLC）发展
  1. Cut a TLC sheet of 8 − 10 cm 8 − 10 cm 8-10cm8-10 \mathrm{~cm} in length, and draw a line at about 1 cm from the bottom with a pencil.
    切一张长度为 8 − 10 cm 8 − 10 cm 8-10cm8-10 \mathrm{~cm} 的薄层色谱板，并在距底部约 1 厘米处用铅笔画一条线。
  2. Put 10 μ L 10 μ L 10 muL10 \mu \mathrm{~L} of the supernatant sample. If several samples are running, keep a 1 cm distance between samples and on each side. Let the solvent evaporate for 5 min (see Note 20).
    加入 10 μ L 10 μ L 10 muL10 \mu \mathrm{~L} 上清液样品。如果同时进行多个样品，样品之间以及样品与侧边保持 1 厘米距离。让溶剂蒸发 5 分钟（参见注释 20）。
  3. Place the TLC sheet into the tilt chamber (at about a 45 ∘ 45 ∘ 45^(@)45^{\circ} angle in relation to the wall chamber).
    将 TLC 薄片放入倾斜室（与壁室呈约 45 ∘ 45 ∘ 45^(@)45^{\circ} 的角度）。
  4. Fill the TLC chamber up to 0.5 cm with mobile phase and close the chamber.
    向 TLC 室中填充 0.5 厘米高的流动相并关闭室盖。
  5. Run the TLC until the mobile phase reaches to the upper side of the TLC sheet ( 0.5 cm before the edge).
    运行 TLC 直到流动相到达 TLC 薄片的上侧（距边缘 0.5 厘米处）。
  6. Dry the sheet for 5 − 10 min 5 − 10 min 5-10min5-10 \mathrm{~min} at room temperature.
    在室温下将薄片干燥 5 − 10 min 5 − 10 min 5-10min5-10 \mathrm{~min} 。
  7. Develop the TLC by spreading 20 % H 2 SO 4 20 % H 2 SO 4 20%H_(2)SO_(4)20 \% \mathrm{H}_{2} \mathrm{SO}_{4} onto the surface with a cotton swab or spraying it.
    使用棉签或喷洒将 TLC 铺展在表面上。
  8. Let it dry and then heat it on a heating plate or in an oven at 100 ∘ C 100 ∘ C 100^(@)C100^{\circ} \mathrm{C} until color development (see Note 21).
    使其干燥，然后在加热板或烤箱中以 100 ∘ C 100 ∘ C 100^(@)C100^{\circ} \mathrm{C} 加热，直到显色（参见注释 21）。

4 Notes  4 注释

  1. The preparation of a Working Cell Bank (WCB) stored at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} is desirable to adequately preserve the strain and standardize the seeding procedure.
    制备存储在 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} 的工作细胞库（WCB）有利于充分保存菌株并标准化接种程序。
  2. Seed a TSA Petri plate and incubate at 31 ∘ C 31 ∘ C 31^(@)C31{ }^{\circ} \mathrm{C} for 24 − 48 h 24 − 48 h 24-48h24-48 \mathrm{~h} to detect potential microbial contaminations.
    在 TSA 培养皿上接种并在 31 ∘ C 31 ∘ C 31^(@)C31{ }^{\circ} \mathrm{C} 孵育 24 − 48 h 24 − 48 h 24-48h24-48 \mathrm{~h} ，以检测潜在的微生物污染。
  3. If necessary, the incubation time can be extended up to 80 h without any negative effect.
    必要时，孵育时间可延长至 80 小时，不会产生任何负面影响。
  4. It is important to avoid wetting the plugs, so you can incubate at 180 rpm if necessary.
    重要的是避免弄湿塞子，因此如有必要可以在 180 rpm 下孵育。
  5. It is important to keep the DO 2 DO 2 DO_(2)\mathrm{DO}_{2} over 30 % 30 % 30%30 \% to guarantee an optimal oxygen supply for growth. An agitation cascade associated to DO 2 DO 2 DO_(2)\mathrm{DO}_{2} level is useful; when DO 2 DO 2 DO_(2)\mathrm{DO}_{2} is going down, the agitation is going up to keep DO 2 DO 2 DO_(2)\mathrm{DO}_{2} over 30 % 30 % 30%30 \%.
    保持 DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 高于 30 % 30 % 30%30 \% 非常重要，以确保生长所需的最佳氧气供应。与 DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 水平相关联的搅拌级联很有用；当 DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 下降时，搅拌会上升以保持 DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 高于 30 % 30 % 30%30 \% 。
  6. To calculate the volume of this solution, subtract the volume corresponding to oil from the final volume ( 3 L ).
    要计算该溶液的体积，从最终体积（3 L）中减去对应于油的体积。
  7. The use of a blender will facilitate the breakdown of the phytosterol crystals, improving the suspension of oil-phytosterol. Place the oil and phytosterol inside the glass vessel and mix with the blender for 2 − 5 min 2 − 5 min 2-5min2-5 \mathrm{~min}.
    使用搅拌机将有助于破碎植物固醇晶体，改善油-植物固醇的悬浮状态。将油和植物固醇放入玻璃容器内，使用搅拌机混合 2 − 5 min 2 − 5 min 2-5min2-5 \mathrm{~min} 。
  8. Once the autoclave sterilization occurs, let the media cool down while applying strong agitation; otherwise, the oil-phytosterol mixture can solidify at the bottom, and it will not be available for M. neoaurum during the bioconversion process.
    自动卡伐灭菌后，在冷却培养基的同时施加强烈搅拌；否则，油-植物固醇混合物可能会在底部凝固，在新鲜链霉菌生物转化过程中将无法使用。
  9. Up until 60 − 70 h 60 − 70 h 60-70h60-70 \mathrm{~h}, the broth has a heterogeneous aspect because of the presence of phytosterol globules. From 60 − 70 h 60 − 70 h 60-70h60-70 \mathrm{~h} onward, the broth takes a more homogeneous appearance, and the DO 2 DO 2 DO_(2)\mathrm{DO}_{2} rises to reach values of about 100 % 100 % 100%100 \% at the end of the bioconversion.
    直到 60 − 70 h 60 − 70 h 60-70h60-70 \mathrm{~h} ，培养液由于存在植物固醇球呈现异质外观。从 60 − 70 h 60 − 70 h 60-70h60-70 \mathrm{~h} 开始，培养液呈现更均质的外观， DO 2 DO 2 DO_(2)\mathrm{DO}_{2} 上升到生物转化结束时约 100 % 100 % 100%100 \% 的值。
  10. If there is a significant difference between the pH values measured offline and online, it is recommended to recalibrate the online pH .
    如果离线和在线测量的 pH 值之间存在显著差异，建议重新校准在线 pH 值。
  11. In this case, the packed cells are located in the oily phase at the top of the tube due to the hydrophobic nature of the mycobacterial cellular surface.
    在这种情况下，由于分枝杆菌细胞表面的疏水性，packed cells 位于试管顶部的油相中。
  12. Phytosterols are not soluble in water, and it is usual to find some phytosterol stuck to the vessel wall or baffles, which is not available for the bioconversion, decreasing the AD bioconversion yield. To minimize this effect, it is advisable to disconnect the engine daily and manually shake the glass vessel, trying to break the unavailable phytosterol free.
    植物甾醇不溶于水，通常会发现一些植物甾醇粘附在容器壁或挡板上，这些植物甾醇不可用于生物转化，从而降低厌氧消化生物转化的产率。为了最大限度地减少这种影响，建议每天断开发动机并手动摇晃玻璃容器，尝试使不可用的植物甾醇脱离。
  13. In this case it is not necessary to mix previously the oil and phytosterol with a blender, because the medium sterilization process occurs with agitation.
    在这种情况下，不需要使用搅拌机预先混合油和植物甾醇，因为介质灭菌过程会进行搅拌。
  14. Apply the pressure test, close the air output, and start to put air inside ( 5 L / min 5 L / min 5L//min5 \mathrm{~L} / \min ) until it reaches a pressure of 1 bar ; then stop the air flow and wait for at least 5 min . The pressure should remain constant during that time.
    按照压力测试要求，关闭气体输出口，开始向内部（ 5 L / min 5 L / min 5L//min5 \mathrm{~L} / \min ）充气，直至压力达到 1 巴；然后停止气流，并等待至少 5 分钟。在此期间，压力应保持恒定。
  15. During the cooling phase of the sterilization process, beware when changing the filter position from “Sterilization” to “Fermentation,” as some foaming may occur. To minimize it, change the filter position when the temperature reaches 60 ∘ C 60 ∘ C 60^(@)C60^{\circ} \mathrm{C}. If temperature is lower, the phytosterol may crystallize inside the air sparger and block the air entry.
    在灭菌过程的冷却阶段，从"灭菌"位置切换到"发酵"位置时要小心，因为可能会出现一些起泡现象。为了最大程度地减少起泡，请在温度达到 60 ∘ C 60 ∘ C 60^(@)C60^{\circ} \mathrm{C} 时更改滤芯位置。如果温度较低，植物固醇可能会在空气曝气器内部结晶并堵塞气体入口。
  16. The bioconversion process at 20 L scale is similar to the one described at 5 L scale, but the third vegetative used to inoculate the 15 L broth has normally more biomass content, and the physical parameters can be slightly different (impeller size, back pressure, oxygen transfer rate, etc.) [22]. At 20 L scale, the heterogeneous aspect of the bioconversion broth changes at 50 − 60 h 50 − 60 h 50-60h50-60 \mathrm{~h} (about 10 − 20 h 10 − 20 h 10-20h10-20 \mathrm{~h} earlier than at 5 L scale), and the bioconversion is completed after 96 − 100 h 96 − 100 h 96-100h96-100 \mathrm{~h}.
    20 L 规模的生物转化过程与 5 L 规模的过程相似，但用于接种 15 L 培养基的第三个营养阶段通常具有更多的生物质含量，且物理参数可能略有不同（搅拌器尺寸、背压、氧气传递率等）[22]。在 20 L 规模下，生物转化培养基的异质性会在 50 − 60 h 50 − 60 h 50-60h50-60 \mathrm{~h} 发生变化（比 5 L 规模提前约 10 − 20 h 10 − 20 h 10-20h10-20 \mathrm{~h} ），并且生物转化在 96 − 100 h 96 − 100 h 96-100h96-100 \mathrm{~h} 后完成。
  17. To take a sample, sterilize with steam the sampling valve for 15 min . Then, discard the first 50 mL of sample and place 50 − 60 mL 50 − 60 mL 50-60mL50-60 \mathrm{~mL} in a sterile container. Finally, sterilize the sampling valve again for 15 min with steam.
    要采样，请用蒸汽对取样阀进行 15 分钟的灭菌处理。然后，丢弃前 50 mL 样品，并将 50 − 60 mL 50 − 60 mL 50-60mL50-60 \mathrm{~mL} 置于无菌容器中。最后，再次使用蒸汽对取样阀进行 15 分钟的灭菌处理。
  18. Butyl acetate retention time is 4.5 min . It could cause confusion and hide some other HPLC chromatogram peaks.
    乙酸丁酯的保留时间为 4.5 分钟。这可能会造成混淆并隐藏一些其他的高效液相色谱图峰。
  19. The use of organic solvents with low boiling point, such as ethyl acetate, is highly recommendable for the sample extraction to be analyzed by TLC. These solvents evaporate quickly once the sample on the TLC sheet is uploaded, avoiding the sample from spreading.
    建议使用低沸点有机溶剂，如乙酸乙酯，用于薄层色谱分析的样品提取。这些溶剂在将样品上传到薄层色谱板后能快速蒸发，避免样品扩散。
  20. For a proper TLC development, put 3 μ L 3 μ L 3muL3 \mu \mathrm{~L} of the sample as a small drop on the line and let the solvent evaporate. Repeat the operation over the same drop until completing the sample loading.
    为了正确进行薄层色谱发展，将 3 μ L 3 μ L 3muL3 \mu \mathrm{~L} 的样品作为小点滴放置在线上，并让溶剂蒸发。重复在同一点滴上进行操作，直到完成样品加载。
  21. It is advisable to include standards of phytosterol and AD of known concentration in the TLC sheet to help identify and quantify the product bands [23].
    建议在薄层色谱板上包含已知浓度的植物甾醇和 AD 标准物，以帮助识别和定量产品条带[23]。

Acknowledgments  致谢

This work was fully supported by grants of the European Union program ERA-IB [MySterI (EIB.12.010)] and ERA CoBioTech for the project Syntheroids. MySterI funding was received through the APCIN call of the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) (PCIN-2013-024-C02-01). Syntheroids funding was received from the European Union’s Horizon 2020 research and innovation program under grant agreement No [722361] and financed through the Center for the Industrial Technology Development (CDTI, Spain) (EXP-00108753/ SERA-20181033). The authors want to thank the European Union programs ERA-IB and ERA CoBioTech, the Spanish Ministry of Economy and Competitiveness (MINECO, Spain), and the Center for the Industrial Technology Development (CDTI, Spain), as well as the MySterI Consortium (INBIOTEC, Pharmins Ltd., University of York, SINTEF, Technische Universität Dortmund, and Gadea Biopharma S.L.) and Syntheroids Consortium (INBIOTEC, Pharmins Ltd., SINTEF, Technische Universität Dortmund and Bionice S.L).
本研究得到了欧盟项目 ERA-IB [MySterI (EIB.12.010)]和 ERA CoBioTech 项目 Syntheroids 的全面支持。MySterI 资金通过西班牙经济和竞争力部（MINECO，西班牙）的 APCIN 呼吁获得（PCIN-2013-024-C02-01）。Syntheroids 资金来自欧盟 Horizon 2020 研究和创新计划，根据拨款协议 No [722361]，并通过工业技术发展中心（CDTI，西班牙）（EXP-00108753/ SERA-20181033）资助。作者感谢欧盟项目 ERA-IB 和 ERA CoBioTech、西班牙经济和竞争力部（MINECO，西班牙）、工业技术发展中心（CDTI，西班牙），以及 MySterI 联盟（INBIOTEC、Pharmins 有限公司、约克大学、SINTEF、多特蒙德工业大学和 Gadea Biopharma 有限公司）和 Syntheroids 联盟（INBIOTEC、Pharmins 有限公司、SINTEF、多特蒙德工业大学和 Bionice 有限公司）。

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