Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients

Two-stage fermentation process was used for lipid production by Lipomyces starkeyi AS 2.1560 in glucose solution without auxiliary nutrients. In the first stage, cells were cultivated in a nutrient-rich medium for propagation. In the second stage, cells were resuspended in glucose solution to achieve high cellular lipid contents. The effects of the inocula age, cell density and initial glucose concentration on lipid production were briefly studied. When high cell density fermentation was performed in a 7-L stirred-tank bioreactor for 40 h using non-sterile glucose solution as carbon source, the biomass, lipid and lipid content reached 104.6 g/L, 67.9 g/L and 64.9%, respectively. More significantly, lipid productivity reached 2.0 g/L h during the initial 16 h-period and 1.6 g/L h for the entire culture. Our results demonstrated that cell propagation and lipid accumulation processes can be spatially separated, allowing further optimization to improve both processes. The two-stage fermentation method should have a great potential to develop more efficient processes to convert renewable materials into biofuel and related products.     

A mutant form of 3-ketosteroid-Δ<Superscript>1</Superscript>-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> ST-095

Mycobacterium neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD), produce very different molar ratios of ADD/AD. The distinct differences were related to the enzyme activity of 3-ketosteroid-Δ¹-dehydrogenase (KSDD), which catalyzes the C_1,2 dehydrogenation of AD to ADD specifically. In this study, by analyzing the primary structure of KSDD_I (from M. neoaurum ST-095) and KSDD_II (from M. neoaurum JC-12), we found the only difference between KSDD_I and KSDD_II was the mutation of Val³⁶⁶ to Ser³⁶⁶. This mutation directly affected KSDD enzyme activity, and this result was confirmed by heterologous expression of these two enzymes in Bacillus subtilis. Assay of the purified recombinant enzymes showed that KSDD_II has a higher C_1,2 dehydrogenation activity than KSDD_I. The functional difference between KSDD_I and KSDD_II in phytosterol biotransformation was revealed by gene disruption and complementation. Phytosterol transformation results demonstrated that ksdd _I and ksdd _II gene disrupted strains showed similar ADD/AD molar ratios, while the ADD/AD molar ratios of the ksdd _I and ksdd _II complemented strains were restored to their original levels. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. Finally, KSDD structure analysis revealed that the Val³⁶⁶Ser mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD. This study provides a reliable theoretical basis for understanding the structure and catalytic mechanism of the Mycobacteria KSDD enzyme.     

Overexpression of cytochrome p450 125 in <Emphasis Type="Italic">Mycobacterium</Emphasis>: a rational strategy in the promotion of phytosterol biotransformation

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD⁺/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD⁺ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD⁺ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD⁺/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.     

Single nucleotide polymorphism analysis for the production of valuable steroid intermediates in <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis>

Objectives

To investigate single nucleotide polymorphism (SNP) in the transformation process of phytosterol to valuable steroid intermediates in three steroid-producing Mycobacterium neoaurum strains using deep sequencing and bioinformation analysis.

Results

The assembled contig sequences from RNA sequencing of strains producing 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA) were analyzed for the presence of putative SNPs for steroid catabolism. 413, 375, and 491 SNPs were detected in the coding domain sequences and non-coding domain sequences of RNA sequencing reads of M. neoaurum strains producing 9OHAD, ADD, and BNA, respectively. Special attention was focused on SNPs associated with genes showing differential expression at proteome level, including the genes for sterol catabolism, glycerol catabolic process, signal transduction systems, transport system and energy metabolism.

Conclusions

The work facilitates the understanding of underlying genetic changes that may be responsible for steroid accumulation in M. neoaurum and is useful for its targeted genetic engineering.

     

Efficient Simultaneous Saccharification and Fermentation of Inulin to 2,3-Butanediol by Thermophilic Bacillus licheniformis ATCC 14580

2,3-Butanediol (2,3-BD) is an important starting material for the manufacture of bulk chemicals. For efficient and large-scale production of 2,3-BD through fermentation, low-cost substrates are required. One such substrate, inulin, is a polydisperse fructan found in a wide variety of plants. In this study, a levanase with high inulinase activity and high pH and temperature stability was identified in Bacillus licheniformis strain ATCC 14580. B. licheniformis strain ATCC 14580 was found to efficiently produce 2,3-BD from fructose at 50°C. Then, the levanase was used for simultaneous saccharification and fermentation (SSF) of inulin to 2,3-BD. A fed-batch SSF yielded 103.0 g/liter 2,3-BD in 30 h, with a high productivity of 3.4 g/liter · h. The results suggest that the SSF process developed with the thermophilic B. licheniformis strain used might be a promising alternative for efficient 2,3-BD production from the favorable substrate inulin.     

表达3-甾酮-△1-脱氢酶降解植物甾醇合成雄甾-1,4-二烯-3,17-二酮

构建分枝杆菌表达载体pMTac并在分枝杆菌Mycobacterium neoaurum JC-12中加强表达甾醇降解过程中的关键酶3-甾酮-△1-脱氢酶(KSDD)以提高雄甾-1,4-二烯-3,17-二铜(ADD)的产量。将p MF41的启动子pACE替换成tac启动子构建载体pMTac,在分枝杆菌中分别表达报告基因绿色荧光蛋白(GFP)和关键酶KSDD,通过GFP亮度和KSDD酶活验证tac启动子在M.neoaurum JC-12中的效果,并发酵验证加强表达KSDD对产物ADD的影响。荧光显微照片表明两个载体均能在M.neoaurum JC-12表达GFP,但tac启动子的效果比pACE强。酶活测定结果为重组菌M.neoaurum JC-12/pMTac-ksdd破碎细胞上清液中KSDD酶活比原始菌提高了6.53倍,比M.neoaurum JC-12/pMF41-ksdd提高了4.36倍。摇瓶发酵显示重组菌M.neoaurum JC-12/pMTac-ksdd ADD的产量比原始菌提高了22.2%,由4.86 g/L提高到5.94 g/L,而AD的产量由0.92 g/L减少到0.17 g/L,降低了81.5%;与M.neoaurum JC-12/p MF41-ksdd比,ADD产量提高了12.7%,AD降低了71.2%。以20 g/L植物甾醇为底物,5 L发酵罐中重组菌M.neoaurum JC-12/pMTac-ksdd的ADD产量达到10.28 g/L。结果表明,构建的新型表达载体pMTac适用于在M.neoaurum JC-12中加强表达关键酶KSDD,而且在M.neoaurum JC-12中过量表达KSDD有助于ADD产量的提高,为目前报道的发酵法利用新金色分枝杆菌降解植物甾醇合成ADD的最高水平。     

Ethanol Production from Colpomenia sinuosa by an Alginate Fermentation Strain Meyerozyma guilliermondii

With the consumption of energy and the spread of COVID-19, the demand for ethanol production is increasing in the world. The industrial ethanol fermentation microbes cannot metabolize the alginate component of macro algae, which affects the ethanol yield. In this research, the ethanol production process from macro algae by an alginate fermentation yeast Meyerozyma guilliermondii, especially the pretreatment process of Colpomenia sinuosa, was studied. At the same time, the experimental design of Box-Behnken was carried out to achieve the optimum fermentation performance. The concentration of KH₂PO₄ (A: 2–6 g.L⁻¹), pH (B: 4–7), reaction time (C: 60–120 h) and temperature (D: 24–34 °C) were variable input parameters. During the ethanol production process, the algae powder was firstly mixed with water at 90 °C for 0.5 h. Later the fermentation culture medium was prepared and then it was fermented by the yeast Meyerozyma guilliermondii to produce ethanol. And the optimal fermentation parameters were as follows: fermentation temperature of 28 °C, KH₂PO₄ dosage of 4.7 g.L⁻¹, initial pH of 6, and fermentation time of 99 h. The ethanol yield reached 0.268 g.g⁻¹ (ethanol to algae), close to the predicted value of model. The generation of alginate lyase during the fermentation of algae was also examined. The highest alginate lyase activity reached 46.42 U.mL⁻¹.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

Characterization of Thermoanaerobacter Glucose Isomerase in Relation to Saccharidase Synthesis and Development of Single-Step Processes for Sweetener Production

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and β-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80°C, pH 7.0 to 7.5; glucogenic amylase, 70°C, pH 5.0 to 5.5; and β-galactosidase, 60°C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and β-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70°C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.     

Coupling the fermentation and membrane separation process for polyamides monomer cadaverine production from feedstock lysine

Nylon is a polyamide material with excellent performance used widely in the aviation and automobile industries, and other fields. Nylon monomers such as hexamethylene diamine and other monomers are in huge demand. Therefore, in order to expand the methods of nylon production, we tried to develop alternative bio‐manufacturing processes which would make a positive contribution to the nylon industry. In this study, the engineered E. coli‐overexpressing Lysine decarboxylases (LDCs) were used for the bioconversion of l‐lysine to cadaverine. An integrated fermentation and microfiltration (MF) process for high‐level cadaverine production by E. coli was established. Concentration was increased from 87 to 263.6 g/L cadaverine after six batch coupling with a productivity of 3.65 g/L‐h. The cadaverine concentration was also increased significantly from 0.43 g cadaverine/g l‐lysine to 0.88 g cadaverine/g l‐lysine by repeated batch fermentation. These experimental results indicate that coupling the fermentation and membrane separation process could benefit the continuous production of cadaverine at high levels.     

Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.     

采用玉米秸秆水解糖和玉米浆发酵生产丁二酸

研究了以玉米秸秆水解糖为碳源,不同氮源条件下琥珀酸放线杆菌Actinobacillus succinogenesSF-9的丁二酸发酵产酸能力。结果表明玉米浆可以替代酵母膏作为丁二酸发酵的廉价氮源。厌氧摇瓶丁二酸发酵单因素试验,得到在初糖浓度50 g/L时,玉米浆的较佳用量为20 g/L。在5 L搅拌罐上,考察了不同初始玉米秸秆水解糖浓度对A.succinogenes SF-9发酵生产丁二酸的影响,结果显示高初始秸秆糖浓度对琥珀酸放线杆菌的生长有抑制作用。采用补料分批发酵,发酵60 h丁二酸的产量达到42.7g/L,丁二酸产率82.7%,生产强度0.81 g/(L·h)。丁二酸的产量和生产强度较分批发酵有明显提高。     

Efficient Open Fermentative Production of Polymer-Grade L-Lactate from Sugarcane Bagasse Hydrolysate by Thermotolerant Bacillus sp. Strain P38

Lactic acid is one of the top 30 potential building-block chemicals from biomass, of which the most extensive use is in the polymerization of lactic acid to poly-lactic-acid (PLA). To reduce the cost of PLA, the search for cheap raw materials and low-cost process for lactic acid production is highly desired. In this study, the final titer of produced L-lactic acid reached a concentration of 185 g·L⁻¹ with a volumetric productivity of 1.93 g·L⁻¹·h⁻¹ by using sugarcane bagasse hydrolysate as the sole carbon source simultaneously with cottonseed meal as cheap nitrogen sources under the open fed-batch fermentation process. Furthermore, a lactic acid yield of 0.99 g per g of total reducing sugars was obtained, which is very close to the theoretical value (1.0 g g⁻¹). No D-isomer of lactic acid was detected in the broth, and thereafter resulted in an optical purity of 100%, which exceeds the requirement of lactate polymerization process. To our knowledge, this is the best performance of fermentation on polymer-grade L-lactic acid production totally using lignocellulosic sources. The high levels of optically pure l-lactic acid produced, combined with the ease of handling and low costs associated with the open fermentation strategy, indicated the thermotolerant Bacillus sp. P38 could be an excellent candidate strain with great industrial potential for polymer-grade L-lactic acid production from various cellulosic biomasses.     

Minimization of Glycerol Production during the High-Performance Fed-Batch Ethanolic Fermentation Process in Saccharomyces cerevisiae, Using a Metabolic Model as a Prediction Tool

On the basis of knowledge of the biological role of glycerol in the redox balance of Saccharomyces cerevisiae, a fermentation strategy was defined to reduce the surplus formation of NADH, responsible for glycerol synthesis. A metabolic model was used to predict the operating conditions that would reduce glycerol production during ethanol fermentation. Experimental validation of the simulation results was done by monitoring the inlet substrate feeding during fed-batch S. cerevisiae cultivation in order to maintain the respiratory quotient (RQ) (defined as the CO₂ production to O₂ consumption ratio) value between 4 and 5. Compared to previous fermentations without glucose monitoring, the final glycerol concentration was successfully decreased. Although RQ-controlled fermentation led to a lower maximum specific ethanol production rate, it was possible to reach a high level of ethanol production: 85 g · liter⁻¹ with 1.7 g · liter⁻¹ glycerol in 30 h. We showed here that by using a metabolic model as a tool in prediction, it was possible to reduce glycerol production in a very high-performance ethanolic fermentation process.     

Efficient co‐production of propionic acid and succinic acid by Propionibacterium acidipropionici using membrane separation coupled technology

To improve the fermentation efficiency of Propionibacterium acidipropionici, a semi‐continuous coupled fermentation process was established to achieve co‐production of propionic acid (PA) and succinic acid (SA). First, the optimal proportion of glucose (Glc) and glycerol (Gl) as a mixed carbon source was determined, and the feeding procedure of Gl was optimized to make more energy flow in the direction of product synthesis. Then, ZGD630 anion exchange resin was used for efficient adsorption of PA, thereby eliminating the feedback inhibition effect of PA. Finally, an efficient, coupled fermentation process of P. acidipropionici characterized by membrane separation and chromatography technology was developed. The concentrations of PA and SA reached 62.22 ± 2.32 and 20.45 ± 1.34 g L⁻¹, with corresponding productivity of 0.43 and 0.14 g L⁻¹ h⁻¹, increased by 65.38% and 48.54%, respectively. Membrane separation coupled fermentation of PA and SA could significantly improve the process economics of P. acidipropionici, and has good prospects for industrial application.     

Production of Higher Alcohols During Indonesian Tapé Ketan Fermentation

A study was made of the higher alcohols (fusel oils) produced during the Indonesian tapé ketan fermentation using Amylomyces rouxii as the principal mold, alone or in combination with yeasts belonging to genera commonly found in the tapé ketan fermentation (Endomycopsis, Candida, and Hansenula). Total fusel oils increased with length of fermentation. Fusel oils detected in the product distillate included isobutanol and isoamyl and active amyl alcohols. No n-propanol was detected. Isobutanol and isoamyl alcohols were formed in the largest amounts. A. rouxii alone produced nearly the same quantity of fusel oils (total production, 275 mg/liter at 192 h) as it did in combination with Endomycopsis burtonii (total production, 292 mg/liter at 192 h).A. rouxii and Endomycopsis fibuliger produced fusel oils totaling 72 mg/liter at 32 h and 558 mg/liter at 192 h. A. rouxii in combination with Candida yeasts produced somewhat more fusel oils, ranging from 590 to 618 mg/liter at 192 h. A. rouxii in combination with Hansenula yeasts produced the least fusel oils, totaling 143 to 248 mg/liter at 192 h. During the first 36 h, production of fusel oils was higher at 30 and 35°C than at 25°C. At 48 h fusel oil production was slightly higher at 30°C than at 35°C. Beyond 48 h, production of fusel oils was higher at 25°C. A. rouxii in combination with Hansenula anomala and Hansenula subpelliculosa produced considerable ethyl acetate, ranging from 145 to 199 mg/liter at 36 h and 354 to 369 mg/liter at 192 h.     

Rational design of a synthetic Entner–Doudoroff pathway for enhancing glucose transformation to isobutanol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Isobutanol as a more desirable biofuel has attracted much attention. In our previous work, an isobutanol-producing strain Escherichia coli LA09 had been obtained by rational redox status improvement under guidance of the genome-scale metabolic model. However, the low transformation from sugar to isobutanol is a limiting factor for isobutanol production by E. coli LA09. In this study, the intracellular metabolic profiles of the isobutanol-producing E. coli LA09 with different initial glucose concentrations were investigated and the metabolic reaction of fructose 6-phosphate to 1, 6-diphosphate fructose in glycolytic pathway was identified as the rate-limiting step of glucose transformation. Thus, redesigned carbon catabolism was implemented by altering flux of sugar metabolism. Here, the heterologous Entner–Doudoroff (ED) pathway from Zymomonas mobilis was constructed, and the adaptation of upper and lower parts of ED pathway was further improved with artificial promoters to alleviate the accumulation of toxic intermediate metabolite 2-keto-3-deoxy-6-phospho-gluconate (KDPG). Finally, the best isobutanol-producing E. coli ED02 with higher glucose transformation and isobutanol production was obtained. In the fermentation of strain E. coli ED02 with 45 g/L initial glucose, the isobutanol titer, yield and average producing rate were, respectively, increased by 56.8, 47.4 and 88.1% to 13.67 g/L, 0.50 C-mol/C-mol and 0.456 g/(L × h) in a shorter time of 30 h, compared with that of the starting strain E. coli LA09.     

Continuous production of biohythane from hydrothermal liquefied cornstalk biomass via two-stage high-rate anaerobic reactors

Background

Biohythane production via two-stage fermentation is a promising direction for sustainable energy recovery from lignocellulosic biomass. However, the utilization of lignocellulosic biomass suffers from specific natural recalcitrance. Hydrothermal liquefaction (HTL) is an emerging technology for the liquefaction of biomass, but there are still several challenges for the coupling of HTL and two-stage fermentation. One particular challenge is the limited efficiency of fermentation reactors at a high solid content of the treated feedstock. Another is the conversion of potential inhibitors during fermentation. Here, we report a novel strategy for the continuous production of biohythane from cornstalk through the integration of HTL and two-stage fermentation. Cornstalk was converted to solid and liquid via HTL, and the resulting liquid could be subsequently fed into the two-stage fermentation systems. The systems consisted of two typical high-rate reactors: an upflow anaerobic sludge blanket (UASB) and a packed bed reactor (PBR). The liquid could be efficiently converted into biohythane via the UASB and PBR with a high density of microbes at a high organic loading rate.

Results

Biohydrogen production decreased from 2.34 L/L/day in UASB (1.01 L/L/day in PBR) to 0 L/L/day as the organic loading rate (OLR) of the HTL liquid products increased to 16 g/L/day. The methane production rate achieved a value of 2.53 (UASB) and 2.54 L/L/day (PBR), respectively. The energy and carbon recovery of the integrated HTL and biohythane fermentation system reached up to 79.0 and 67.7%, respectively. The fermentation inhibitors, i.e., 5-hydroxymethyl furfural (41.4–41.9% of the initial quantity detected) and furfural (74.7–85.0% of the initial quantity detected), were degraded during hydrogen fermentation. Compared with single-stage fermentation, the methane process during two-stage fermentation had a more efficient methane production rate, acetogenesis, and COD removal. The microbial distribution via Illumina MiSeq sequencing clarified that the biohydrogen process in the two-stage systems functioned not only for biohydrogen production, but also for the degradation of potential inhibitors. The higher distribution of the detoxification family Clostridiaceae, Bacillaceae, and Pseudomonadaceae was found in the biohydrogen process. In addition, a higher distribution of acetate-oxidizing bacteria (Spirochaetaceae) was observed in the biomethane process of the two-stage systems, revealing improved acetogenesis accompanied with an efficient conversion of acetate.

Conclusions

Biohythane production could be a promising process for the recovery of energy and degradation of organic compounds from hydrothermal liquefied biomass. The two-stage process not only contributed to the improved quality of the gas fuels but also strengthened the biotransformation process, which resulted from the function of detoxification during biohydrogen production and enhanced acetogenesis during biomethane production.

     

Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01

Mycobacterium neoaurum NwIB-01 exhibits powerful ability to cleave the side chain of soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). The difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. To enhance ADD quantity in products, 3-ketosteroid Δ¹-dehydrogenase genes (kstD _M and kstD _A) were obtained from M. neoaurum NwIB-01 and Arthrobacter simplex respectively. Using replicating vector pMV261, kstD _M and kstD _A were overexpressed in M. neoaurum NwIB-01. For foreign gene stable expression, the integration vector pMV306 was used for kstD _M/kstD _A overexpression and the relevant sequences of promoter and kanamycin antibiotic resistance gene sequences were amplified by PCR to verify plasmid integrity. The resultant plasmid and mutant strain were verified and the kstD augmentation mutants were good ADD-producing strains. The ADD producing capacity of NwIB-04 and NwIB-05 was 0.1401 and 0.1740 g/l (cultured in shake bottles with 0.4 g/l phytosterols), and the molar ratio of ADD in products was 98.34 and 98.60 %, respectively. This study on the manipulation of the main kstD_M gene in Mycobacterium sp. provides a feasible way to achieve excellent phytosterol-transformation strains with high product purity.