Towards Biorefinery Production of Microalgal Biofuels and Bioproducts: Production of Acetic Acid from the Fermentation of <i>Chlorella</i>sp. and <i>Tetraselmis suecica</i>Hydrolysates

Successful commercialization of microalgal bio-industry requires the design of an integrated microalgal biorefinery system that facilitates the co-production of biofuels, high-value products and industrial chemicals from the biomass. In this study, we investigated the use of sugar hydrolysate obtained from enzymatic saccharification of microalgal biomass (Chlorella sp. and T. suecica) as fermentation feedstock to produce industrially important chemicals, in particular acetic acid and butyric acid. By using hydrolysate with low sugar content as substrate for the anaerobic fermentation (1.5 - 2.4 g/L), we were able to prevent the bacterium C. saccharoperbutylacetonicum from activating its solventogenesis pathway. As a result, the fermentation process generated a product stream that was dominated by organic acids (acetic acid and butyric acid) rather than solvents (butanol, ethanol and acetone). Acetic acid constituted up to 92 wt% of Chlorella’s fermentation products and 80 wt% of T. suecica’s fermentation products. For T. suecica, the fermentation consumed almost all of the sugar available in the hydrolysate (up to 92% of initial sugar) and produced a reasonable yield of fermentation products (0.08 g fermentation products/g sugar). The Gompertz equation was successfully used to predict the formation kinetics of acetic acid and other fermentation products across both species. The results in the study demonstrate the production of industrially important chemicals, such as acetic acid and butyric acid, from the fermentation of microalgal sugar. The process described in the study can potentially be used as a value-adding step to generate biochemicals from cell debris in an integrated microalgal biorefinery system.

Formation mechanisms of ethyl acetate and organic acids in Kluyveromyces marxianus L1-1 in Chinese acid rice soup

This study aims to explore the formation mechanism of ethyl acetate and organic acids in acid rice soup(rice-acid soup)inoculated with Kluyveromyces marxianus L1-1 through the complementary analysis of transcriptome and proteome.The quantity of K.marxianus L1-1 varied significantly in the fermentation process of rice-acid soup and the first and third days were the two key turning points in the growth phase of K.marxianus L1-1.Importantly,the concentrations of ethyl acetate,ethanol,acetic acid,and L-lactic acid increased from day 1 to day 3.At least 4231 genes and 2937 proteins were identified and 610 differentially expressed proteins were annotated to 30 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways based on the analysis results of transcriptome and proteome.The key genes and proteins including up-regulated alcohol dehydrogenase family,alcohol O-acetyltransferase,acetyl-CoA C-acetyltransferase,acyl-coenzyme A thioester hydrolase,and down-regulated aldehyde dehydrogenase family were involved in glycolysis/gluconeogenesis pathways,starch and sucrose metabolism pathways,amino sugar and nucleotide sugar metabolism pathways,tricarboxylic acid(TCA)cycle,and pyruvate metabolism pathways,thus promoting the formation of ethyl acetate,organic acids,alcohols,and other esters.Our results revealed the formation mechanisms of ethyl acetate and organic acids in rice-acid soup inoculated with K.marxianus L1-1.

Screening of Acetic Acid Producing Microorganisms from Decomposed Fruits for Vinegar Production

Acetic acid bacteria capable of growing at 30&#176C - 37&#176C were collected from various decomposed fruits available in Bangladeshi local markets in order to assess their suitability for vinegar production. Initially, 42 microorganisms were isolated from decomposed fruits like grapes, mangoes, pineapples, oranges, safeda etc. during summer when temperature reaches up to 37&#176C. Then their growths were checked in YPG medium containing various ethanol concentrations at different time point at 37&#176C. From the preliminary screening, 15 Gram negative bacterial isolates have produced halos or yellow zone around the colonies on YPG agar plate at 37&#176C which indicated acetic acid production capability by those bacteria. Furthermore, acetic acid production rates were determined by titration method and about 3 - 6.9 gm/100ml acetic acid were estimated by using 4% ethanol at 37&#176C by shaking culture for 3 days. Several biochemical analysis revealed that our collection contained huge amount of acetic acid producing bacteria and some of them could be potential candidates for vinegar production.

Immobilization of trophic anaerobic acetogen for semi-continuous syngas fermentation

The massive consumption of fossil energy force s people to find new source s of energy.Syngas fermentation has become a hot research field as its high potential in renewable energy production and sustainable development.In this study,trophic anaerobic acetogen Morella thermoacetica was successfully immobilized by calcium alginate embedding method.The ability of the immobilized cells on production of acetic acid through syngas fermentation was compared in both airlift and bubble column bioreactors.The bubble column bioreactor was selected as the better type of bioreactor.The production of acetic acid reached 32.3 g·L^(-1) in bubble column bioreactor with a space-time yield of 2.13 g·L^(-1)·d^(-1).The immobilized acetogen could be efficiently reused without significant lag period,even if exposed to air for a short time.A semi-continuous syngas fermentation was performed using immobilized cells,with an average space-time acetic acid yield of 3.20 g·L^(-1)·d^(-1).After 30 days of fermentation,no significant decrea se of the acetic acid production rate was observed.

The Phytochemical and Antioxidant Characteristics of Fermented Jackfruit （Artocarpus heterophyllus L.） Leaves Using Single and Mixed Starter Culture

The phytochemical and antioxidant properties of fermented jackfruit leaves, using yeast and acetic acid bacteria, individually or in combination during 6 days of fermentation were investigated in the present study. Changes in pH, total reducing sugar, ethanol, acetic acid, total phenolics and flavanoids content were examined. A number of antioxidant activities such as ferric reducing antioxidant power （FRAP） and DPPH free radical scavenging activities were comparatively tested to determine the differences in the respective properties during the fermentation process. Results obtained demonstrated that there were differences in the phytochemical and antioxidant properties of fermented jackfruit leaves, depending on the fermentation starters. Fermented leaves using acetic acid bacteria alone, exhibited higher total reducing sugar （6.89 mg glucose/mL） compared to others. The ferric-reducing activities of fermented jackfruit leaves showed an increasing trend in the yeast fermentation process （5.96-10.12 mg ascorbic acid equivalent/mL）. Generally, all extracts of fermented jackfruit leaves exhibited stronger DPPH radical scavenging activity except in the total phenolics and flavonoids content, whereby it showed a decrease trend throughout the fermentation process.

重组木聚糖酶的高密度发酵及其定向制备低聚木糖

为提高酶水解杨木木聚糖定向转化低聚木糖的效率,同时降低酶法制备低聚木糖的成本,围绕重组大肠杆菌(PET-PdoXyn10A-DE3)高密度发酵制备拟杆菌来源木聚糖酶PdoXyn10A的诱导表达条件和酶水解制备低聚木糖的工艺条件开展了研究。考察了在5 L发酵罐水平下诱导剂浓度、诱导光密度(OD_(600))值、诱导温度和诱导pH值等因素对PET-PdoXyn10A-DE3产重组木聚糖酶PdoXyn10A的影响,反应温度、pH值对玉米芯木聚糖酶水解过程中酶活力的影响,以及酶添加量、酶水解时间对杨木乙酸水解液制备低聚木糖的影响。研究结果表明:在诱导剂浓度0.25 mmol/L、诱导OD_(600)值55、诱导温度34℃和诱导pH值7.0的最佳诱导条件下,经高密度发酵重组酶酶活力可达362.67 U/mL,重组大肠杆菌生物量可达28.83 g/L。酶水解制备低聚木糖的最佳条件为酶反应温度40℃、pH值5.2、酶添加量12 U/mL和反应时间6 h,此条件下低聚木糖的量为3.21 g/L,其中木二糖、木三糖、木四糖、木五糖和木六糖的量分别为1.09、 0.97、 0.84、 0.18、 0.13 g/L,木糖为1.82 g/L。

厨余垃圾厌氧发酵制备乙酸技术及应用进展

我国垃圾分类全面实施后厨余垃圾产量骤增,目前采用主流的厌氧消化工艺因沼气利用率低、产品附加值低、沼液处理成本高等问题导致项目经济效益不佳,厨余垃级资源化利用技术亟需升级。有机物厌氧发酵制备的有机酸工业用途广泛,其中乙酸是医药、化工等行业的基础原料或者重要中间体,近年来由于能源危机、原材料稀缺以及环境问题促使生物基乙酸需求量日益增加,厨余垃圾发酵制备乙酸为有机固废高值化利用和能源绿色低碳转型提供新出路。通过梳理厨余垃圾厌氧发酵产乙酸过程及其生物代谢机制,阐述不同参数条件对乙酸产量的影响,分析探索提高乙酸产量的方法,为厨余垃圾厌氧产乙酸实际工程应用推广提供参考。

基于高通量测序分析山西老陈醋醋酸发酵过程中细菌群落的演替规律

以山西老陈醋醋醅为研究对象,通过高通量测序技术,探究其在发酵过程中细菌群落的多样性和演替规律。结果表明,山西老陈醋醋酸发酵过程中在门的水平上主要有厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)等7个菌门,其中厚壁菌门的相对丰度随发酵进行呈上升趋势,至发酵末期,其相对丰度甚至达到98%以上,在整个醋酸发酵阶段处于主导地位。在属水平,参与山西老陈醋醋酸发酵阶段的主要细菌有乳杆菌属(Lactobacillus,57.4%~66.4%)、醋酸菌属(Acetobacter,3.8%~27.3%)、链球菌属(Streptococcus,0.3%~3.0%)、魏斯氏菌属(Weissella,0.2%~1.8%)、芽孢杆菌属(Bacillus,0.1%~1.2%)、假单胞菌属(Pseudomonas,0%~1.8%)等,其中乳杆菌属(Lactobacillus)和醋酸菌属(Acetobacter)的细菌占主导优势,发酵前5 d二者的相对丰度之和高达90%左右。发酵前期的醋醅样品中并未检测到假单胞菌属(Pseudomonas),随着醋酸发酵的进行其在第5 d的样本中出现,并在第7 d达到峰值(1.1%~3.4%)。山西老陈醋醋酸发酵阶段采用“套醪接种”,不同发酵批次的细菌群落结构和变化情况基本相同,所共有的微生物物种占比总数的70%以上,说明山西老陈醋固态酿造工艺的稳定性。

液态发酵法酿造豆渣食醋工艺研究

为增加豆渣的利用率和附加值,以豆渣为主要原料,通过液态发酵法酿造一款豆渣食醋,并探索其酒精发酵、醋酸发酵的最佳工艺条件。首先以酒精度为评价指标,通过单因素试验和正交试验对酒精发酵工艺进行优化。其次,以最佳酒精发酵工艺为基础,以食醋的感官评分和总酸含量为评价指标,通过单因素试验和响应面试验对醋酸发酵工艺进行优化。结果表明,以180 mL纯净水为基准,最佳酒精发酵工艺为豆渣干粉添加量14 g、蔗糖添加量6 g、酵母菌接种量0.4%、酒精发酵温度32℃、酒精发酵时间6 d;以100 mL酒精滤液为基准,最佳醋酸发酵工艺为醋酸菌接种量3.85%、醋酸发酵温度29.5℃、醋酸发酵时间5 d。此工艺条件下豆渣食醋的感官评分为83.2分,总酸含量为5.05 g/dL,可溶性无盐固形物含量为3.5 g/dL,氨基酸态氮含量为122.4 mg/dL,菌落总数和大肠菌群均符合国家标准。豆渣食醋成品呈琥珀色,澄清透亮,质地均匀,酸味适中,带有浓郁的醋香和豆渣特有的豆香味,营养价值较高。

多菌种混合发酵神农架猕猴桃果醋的工艺优化及风味分析

以神农架猕猴桃为原料,通过多菌种混合发酵制备猕猴桃果醋。通过单因素及正交试验优化其酒精、醋酸发酵工艺条件,采用氨基酸分析仪及气质联用(GC-MS)技术分别分析果醋的氨基酸、挥发性风味物质组成。结果表明,最佳酒精发酵工艺为:酵母菌接种量0.9%,乳酸菌接种量7%,初始可溶性固形物19°Bx,发酵温度27℃。在此优化条件下,酒精度为(8.82±0.57)%vol,乳酸含量为(12.15±0.73)mg/mL。最佳醋酸发酵工艺为:发酵时间8 d,醋酸菌接种量12%,初始酒精度7%vol,发酵温度32℃。在此优化条件下,醋酸含量为(4.34±0.17)g/100 mL,感官评分为80分。多菌种混合发酵猕猴桃果醋共检出15种氨基酸,包含7种必需氨基酸;共检出37种挥发性风味物质,包括酯类17种,酸类5种,醛类2种,酮类5种,苯类2种,醇类4种,酚类1种,其他类1种。综上,多菌种混合发酵有利于促进氨基酸和挥发性风味物质的形成,从而提升果醋的品质。

醋酸发酵阶段添加不同类型高温大曲对食醋风味品质的影响

该研究将白曲、黄曲、黑曲3种不同类型的高温大曲拌入醋醅进行醋酸发酵,制备固态发酵食醋。采用HPLC和顶空固相微萃取结合气相色谱质谱联用(headspace-solid phase microextraction-gas chromatography-mass spectrometry,HS-SPME-GC-MS)技术,结合正交偏最小二乘判别分析(orthogonal partial least squares-discriminant analysis,OPLS-DA)比较成品醋中有机酸和挥发性化合物的组成差异。结果表明,在醋酸发酵阶段添加3种不同类型的高温大曲均能够提高成品醋的总酸和有机酸含量,主要表现为乳酸和乙酸含量的提高,添加白曲的提升效果最佳;同时,HS-SPME-GC-MS显示3种不同类型高温大曲的添加均能丰富成品醋的挥发性风味,OPLS-DA筛选出38种具有重要差异的挥发性化合物,并且添加黄曲和黑曲的成品醋的挥发性化合物与对照组成品醋的差异更明显;此外,感官评价表明醋酸发酵阶段添加高温大曲能改善成品醋的风味品质,添加黄曲的效果最佳。该研究证实了在醋酸发酵阶段添加3种不同类型的高温大曲可以改善固态发酵食醋的风味品质,为食醋产品的开发和风味品质改良提供参考。

板栗醋发酵工艺优化及挥发性成分分析

为提高板栗资源利用率,开发板栗深加工产品,以板栗为原料,通过酒精和醋酸发酵,研究开发一款新型板栗醋。以感官评价和酸度的综合评分为评价指标,采用单因素和正交试验对板栗醋进行工艺优化。结果表明,酒精发酵优化后工艺条件为酒精发酵温度22℃、酵母菌添加量0.04%、初始糖度17°Brix、酒精发酵时间5 d;醋酸发酵优化后工艺条件为醋酸发酵温度31℃、果醋菌添加量0.04%、装液量40%、转速170 r/min、醋酸发酵时间8 d,优化后板栗醋总酸20.18 g/L,具有黄色透明质感、板栗果香,味道醇厚。采用顶空固相微萃取(headspace solid phase microextraction,HS-SPME)结合气质联用(gas chromatography-mass spectrometry,GC-MS)测定板栗醋3个发酵阶段的挥发性成分并探究其变化,结果表明,板栗醋3个发酵阶段挥发性成分的含量及种类有明显差异,在板栗浆、酒精发酵和醋酸发酵阶段时,挥发性成分含量最高的分别为乙酸(15.36%)、乙醇(30.26%)和乙酸(41.13%)。

枸杞茶醋醋酸发酵工艺优化及抗氧化活性研究

该研究以枸杞芽茶和玉米须为原料,采用巴氏醋杆菌(Acetobacter pasteurianus)AC-2005发酵制备枸杞茶醋,以总酸含量为评价指标,通过单因素试验及正交试验优化枸杞茶醋醋酸发酵工艺,对其理化指标进行测定,并对其发酵前后的功能活性成分和抗氧化活性变化进行分析。结果表明,枸杞茶醋的最优醋酸发酵工艺为初始酒精度9%vol,巴氏醋杆菌AC-2005接种量10%,发酵温度30℃,初始pH值3.5。在此优化条件下,枸杞茶醋的总酸含量为7.10 g/100 mL,pH值为3.56,还原糖含量为1.70 g/100 mL,可溶性固形物含量为9.28 g/100 mL,其理化指标符合相关标准要求。与发酵前相比,枸杞茶醋中的总酚(2.27 mg/mL)和总黄酮含量(0.94 mg/mL)显著升高(P<0.05),甜菜碱含量(2.30 mg/mL)无显著变化(P>0.05),类胡萝卜素含量(0.62 mg/mL)显著降低(P<0.05);抗氧化能力显著升高(P<0.05),1,1-二苯基-2-三硝基苯肼(DPPH)、2,2’-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)自由基清除率和铁离子还原/抗氧化能力(FRAP)分别为20.27%、12.71%和11.68%。

高酸度醋补料发酵工艺优化

高酸度醋的酿造主要采用液态深层发酵技术,高酸度醋有利于储存和运输,但目前高酸度醋的发酵工艺仍不完善。该研究利用福林斯自吸式深层发酵装置,以酿造酸度为9 g/100 mL醋的醋酸菌作为发酵菌株,通过对发酵工艺的调控酿造高酸度醋。结果表明,以14%vol酒精度发酵液进行高酸度醋的酿造,经单因素及响应面法试验得到最佳的发酵工艺条件为:采用直接补料法进行补料,补料时间2.5 h,通气量0.12 m^(3)/(m^(3)·min),营养盐添加量1.7‰,营养盐在补料时加1.3‰+罐内发酵液酒精度降至3.5%vol~2.5%vol时加0.4‰。在此优化条件下,高酸度醋的总酸含量为12.62 g/100 mL,发酵周期为34.06 h,产酸速率为0.12 g/(100 mL·L·h),乙醇转酸率为82.85%。

不同酿造工艺对山西老陈醋乙偶姻及川芎嗪含量的影响

为改善生产工艺,提高老陈醋品质,该研究以醋醅为研究对象,分别考察了传统、机械醋酸发酵过程中酒精度、总酸、乙偶姻、川芎嗪的变化及传统、机械熏醅方式对乙偶姻和川芎嗪含量的影响。结果表明,在传统醋酸发酵工艺条件下,发酵结束时,醋醅酒精度、总酸、乙偶姻、川芎嗪含量分别为0.37%vol、4.85 g/100 mL、706.67 mg/L、0.38 mg/L;在机械醋酸发酵工艺条件下,发酵结束时,酒精度、总酸、乙偶姻、川芎嗪含量分别为0.75%vol、4.25 g/100 mL、2 933.33 mg/L、1.33 mg/L。在传统醋酸发酵工艺条件下,发酵结束时,传统、机械熏醅工艺醋醅乙偶姻、川芎嗪的含量分别为200.23 mg/L、3.15 mg/L,10.45 mg/L、11.18 mg/L;在机械醋酸发酵工艺条件下,发酵结束时,传统、机械熏醅工艺醋醅乙偶姻、川芎嗪的含量分别为1 666.63 mg/L、4.73 mg/L,137 mg/L、17.63 mg/L。综合考虑,选用机械醋酸发酵、机械熏醅工艺方式酿造食醋。

重组大肠杆菌产耐热α-L-鼠李糖苷酶高密度发酵条件的优化

为了减少乙酸的生成,提高重组大肠杆菌生产耐热α-L-鼠李糖苷酶的产量,考查了指数流加培养、两阶段指数流加培养、诱导温度、诱导pH对重组大肠杆菌发酵生产耐热α-L-鼠李糖苷酶(TstRhaA)过程中乙酸含量和酶产量的影响。确定最佳发酵工艺:诱导前比生长速率0.2 h^(-1),诱导后比生长速率0.18 h^(-1),诱导温度33℃,pH 7.2。在最佳发酵工艺条件下,乙酸浓度始终保持在0.8 g/L以下,菌体干重(DCW)最大为69.6 g/L,最高酶活力为589.0 U/mL,是摇瓶发酵的23.4倍,实现了重组大肠杆菌高密度发酵生产TstRhaA。研究结果表明,调节大肠杆菌高密度发酵过程中乙酸含量在合适范围内,能够实现TstRhaA的高效生产,这对其他酶的高密度表达具有重要指导意义。

软枣猕猴桃苹果复合果醋发酵工艺的优化

以软枣猕猴桃和苹果为原料,通过酵母菌酒精发酵和醋酸菌醋酸发酵获得软枣猕猴桃和苹果的复合果醋饮料。以总酸和感官评分作为综合评定指标,通过单因素试验和正交试验优化了软枣猕猴桃苹果复合果醋的醋酸发酵工艺。结果表明,适宜的醋酸发酵工艺为醋酸菌添加量0.3%,装液量150 mL(250 mL三角瓶),发酵时间9 d,发酵温度28℃。获得的复合果醋总酸质量浓度为29.54 g/L,感官评分为92分,酸度适中、果香浓郁,为软枣猕猴桃的开发利用提供了一条新途径。

添加药食同源物质对功能型葡萄醋饮发酵过程的影响及其工艺优化

采用优良醋酸杆菌XC,以葡萄酒为原料,对不同发酵时间下酒精度、发酵温度、种子液接种量及通风量转速进行了单因素醋酸发酵实验。通过正交试验,优化确定葡萄醋发酵工艺为:酒精度6%vol、接种量8%、发酵温度32℃、通风量180r/min,该条件下有挥发酸最大值53.76g/L(以乙酸计)。同时,选取山楂、乌梅、陈皮、紫苏叶、茯苓等7味药食同源药材配伍炮制方剂,与葡萄酒混合进行醋酸发酵,得到一款功能型葡萄醋,其总黄酮、总多酚含量较葡萄原醋均有明显提升,总酸含量为69.90g/L(以酒石酸计)。功能型葡萄醋口感酸甜、葡萄果香突出,药香协调、口感柔和,为新型健康食品的稳定发展提供理论依据。

三华李-木瓜复合果醋的发酵工艺研究

以三华李和木瓜为主要原料,对三华李-木瓜复合果醋的发酵工艺进行优化研究。通过单因素试验探究初始酒精度、果醋菌添加量、发酵温度和发酵时间对复合果醋发酵的影响,在此基础上以总酸为响应值,采用Box-benhnken响应面法进一步优化醋酸发酵工艺。结果表明,三华李-木瓜复合果醋的最佳发酵工艺条件为初始酒精度9%(V/V),果醋菌添加量3%,发酵温度34℃,发酵时间11 d。在此工艺条件下,三华李-木瓜复合果醋的总酸含量达5.17 g/100 mL,果醋外观呈棕红色,澄清透亮、酸味柔和、风味独特。

Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB

The production of butyric acid by Clostridium butyricum ZJUCB at various pH values was investigated. In order to study the effect of pH on cell growth, butyric acid biosynthesis and reducing sugar consumption, different cultivation pH values ranging from 6.0 to 7.5 were evaluated in 5-L bioreactor. In controlled pH batch fermentation, the optimum pH for cell growth and butyric acid production was 6.5 with a cell yield of 3.65 g/L and butyric acid yield of 12.25 g/L. Based on these results, this study then compared batch and fed-batch fermentation of butyric acid production at pH 6.5. Maximum value (16.74 g/L) of butyric acid concentration was obtained in fed-batch fermentation compared to 12.25 g/L in batch fermentation. It was concluded that culti- vation under fed-batch fermentation mode could enhance butyric acid production significantly (P<0.01) by C. butyricum ZJUCB.