Crude Fermentation Broth Research Articles

A closed-loop strategy for on-site production of saccharolytic enzymes for lignocellulose biorefinery using internal lignocellulosic hydrolysates

The cost-effective production of lignocellulose-saccharolytic enzymes is crucial for the bioconversion of plant biomass into fuels and chemicals. The integrated mode for producing these enzymes using lignocellulosic feedstocks as natural inducers has a cost advantage; however, enzyme production level is limited because of the solid-state and complex composition of the raw materials. This study aimed to establish an innovative process for the integrated production of lignocellulolytic enzymes using lignocellulosic hydrolysate as the carbon source. The cellulase-producing workhorse Trichoderma reesei was reprogrammed by combinatorial engineering of transcription factors and the introduction of heterologous β-glucosidases. This enabled inducer-independent production of a complete lignocellulose-saccharolytic enzyme cocktail using lignocellulose-derived monosaccharides as sole carbon source. Via continuous feeding of the enzymatic hydrolysate of pretreated corn stover, crude enzymes with filter paper enzyme activity of 60.4 U/ml and β-glucosidase activity of 503 U/ml were produced. Crude fermentation broth can be used directly to prepare hydrolysates, enabling nex-round enzyme production by recycling 3.1% of the hydrolysate. These results demonstrate a closed-loop strategy for integrated enzyme production using internally produced lignocellulosic sugars from biorefinery plants.

Aqueous two-phase extraction to separate alpha-ketoglutaric acid from fermentation broth by combining crystallization

Alpha-ketoglutaric acid (α-KG) is a crucial intermediate with broad application prospects. However, the downstream separation of α-KG from fermentation broth with low concentration and complex components has become the bottleneck of large-scale production. In this study, α-KG was separated from the actual fermentation broth by aqueous two-phase system (ATPS) and crystallization. The optimal ATPS composed of acetone/(NH4)2SO4 was selected by studying the phase diagram and α-KG distribution behavior in different organic solvents and salts/sugars. Under the optimized conditions, the purity of α-KG products from crude fermentation broth reached 98.08%, and the one-pass extraction efficiency was 90.52%. The removal of cell debris and soluble proteins increased with the increase of acetone and (NH4)2SO4 concentrations, with the removal ratios reaching up to 99.44% and 86.54%. In addition, 92.56% of the glucose was distributed in the aqueous phase. Although the α-KG fermentation broth is more complex than the whole-cell biotransformation, impurities in the crude fermentation broth could be efficiently removed and a higher α-KG distribution coefficient was obtained. Meanwhile, the recovered (NH4)2SO4 could be reused for fermentation medium. This strategy provides an economical and effective method combined with upstream biosynthesis to recover α-KG and other bioproducts from crude fermentation broth.

Strain evolution and novel downstream processing with integrated catalysis enable highly efficient coproduction of 1,3-propanediol and organic acid esters from crude glycerol.

Bioconversion of natural microorganisms generally results in a mixture of various compounds. Downstream processing (DSP) which only targets a single product often lacks economic competitiveness due to incomplete use of raw material and high cost of waste treatment for by-products. Here, we show with the efficient microbial conversion of crude glycerol by an artificially evolved strain and how a catalytic conversion strategy can improve the total products yield and process economy of the DSP. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72, and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The coproduction of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for the simultaneous production of three appealing products from the crude glycerol fermentation broth, which greatly improve the process economy and ecology.

Enantioselective resolution of (±)-1-phenylethyl acetate using the immobilized extracellular proteases from deep-sea Bacillus sp. DL-1

Bacillus sp. DL-1 was isolated from the deep sea of the Western Pacific Ocean and behaved very good resistance to NaCl. The extracellular proteases of Bacillus sp. DL-1 were found to exhibit excellent enantioselectivity for the kinetic resolution of (±)-1-phenylethyl acetate. To improve the stability of the enzyme, the immobilized extracellular proteases were preparated by using 60 g kieselguhr and 1-L crude fermentation broth containing extracellular proteases of Bacillus sp. DL-1, shaking at 25 °C, 200 r/min for 10 h. Every gram of kieselguhr adsorbed 25.7 mg extracellular protease and the enzymatic activity recovery was 79.86%. The immobilized proteases preserved about 35.8% of its activity after 6 repeated uses, which were also used as biocatalyst to asymmetrically hydrolyse (±)-1-phenylethyl acetate for the preparation of (R)-1-phenylethanol and (S)-1-phenylethyl acetate with high optical purities. The effects of pH, temperature, enzyme concentration, substrate concentration, reaction time and additives (metal ions/surfactants) on the resolution were investigated by single factor experiments. Under the optimal reaction conditions (10 mM (±)-1-phenylethyl acetate, 40 mg/mL immobilized extracellular proteases, pH 7.5 (Tris-HCl), 5% (v/v) methanol and 45 °C for 2 h), (R)-1-phenylethanol was generated with the e.e.p being > 97%, and the yield being 53%, respectively. Analogously, under the optimal reaction conditions (10-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.0 (PB), 5% (v/v) DMSO and 35 °C for 1.5 h), (S)-1-phenylethyl acetate was generated with the e.e.s being over 99% and the yield being 79%, respectively. Compared with the extracellular proteases from Bacillus sp. DL-2, the immobilized extracellular proteases from Bacillus sp. DL-1 exhibited higher hydrolytic activity and could asymmetrically hydrolyse (±)-1-phenylethyl acetate by using higher substrate concentrations, shorter reaction times to obtain higher yields. Notably, the extracellular proteases of Bacillus sp. DL-1 were demonstrated to behave the same enantio-preference as those of most other reported esterases/lipases. Proteases from deep-sea Bacillus sp. DL-1 are promising biocatalysts for the synthesis of valuable chiral chemicals.

Application of rotating packed bed for in‐line aroma stripping from cell slurry

AbstractBACKGROUNDNowadays, biotechnological production receives increasing interest as an alternative source of natural aromas. Unfortunately, especially for hydrophobic and semi‐volatile aromas, the heterogeneous product partitioning between all phases present in fermentation makes recovery challenging. Additionally, when an aroma displays an inhibitory effect on the production microorganism, product removal during fermentation is recommendable. In‐line aroma stripping offers an elegant way to deal with such challenges. This study reports the use of rotating packed bed (RPB) technology for the intensification of stripping of α‐ionone, a key aroma of raspberry, from a model fermentation slurry containing Saccharomyces cerevisiae cells in a concentration of 250 g‐CWW L−1.RESULTSThroughout all experimental investigations, yeast cells were robust towards both the chemical stress from aroma exposure at a concentration of up to 400 mg L−1 and the mechanical stress from peripheral equipment and rotation of up to 2750 rpm, as a maximum of 11.3 ± 0.5% disrupted cells were measured during continuous processing in an RPB. An increase in the rotation speed led to an enhanced transfer of α‐ionone from the fermentation slurry to the gaseous phase.CONCLUSIONSRPB technology is found to be promising for the intensification of in‐line stripping of biotechnologically produced aromas from crude fermentation broth without cell separation. The use of subsequent RPBs equipped with custom packings and flexibly adjustable rotation speed displays a holistic aroma recovery process supporting the way to commercial competitiveness of biotechnological aromas. © 2020 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Downstream separation and purification of bio-based alpha-ketoglutaric acid from post-fermentation broth using a multi-stage membrane process

In this study, a multi-stage membrane process, assisted by vacuum evaporation and crystallization, for recovery of bio-based alpha-ketoglutaric acid from the actual post-fermentation broth was designed and investigated. In the first part of this study, pre-treatment of crude fermentation broth (centrifugation-ultrafiltration-nanofiltration) was carried out to remove biomass, proteins, sugars, part of inorganic ions and color compounds. The commercial ceramic UF membrane (15 kDa) and nanofiltration ceramic membrane (200 Da or 450 Da) were applied. Electrodialysis with a bipolar membrane was proposed for separation of ionic compounds and simultaneous electro-acidification to the acid form. During bipolar membrane electrodialysis carried out under acidic conditions, it was possible to remove close to 90 % of alpha-ketoglutaric acid. Moreover, the migration of other acids present in the fermentation broth (lactic and acetic) was significantly limited. The calculated specific energy consumption was low and equal to 0.6 kW h/kg. The final purification using crystallization assisted vacuum evaporation allowed obtaining alpha-ketoglutaric acid in solid form. Analysis of the final product showed that the proposed method of alpha-ketoglutaric acid recovery gives the acid of high purity equal to 94.8 %. Furthermore, the presented results have practical relevance and may in future be the basis for the development of separation technologies of alpha-ketoglutaric acid from the fermentation broth on industrial scale.

Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate Synthesis

Cinnamyl acetate has a wide application in food industries. This work focuses on cinnamyl acetate synthesis with a novel esterase. estGUZ753, an esterase gene from Geobacillus uzenensis DSMZ 13551, was first cloned and expressed in Pichia pastoris KM71. The optimal activity of EstGUZ753 towards p-NP caprylate was at pH 8.0 and 70 °C, and the half-life at 70 °C was 28 h. Furthermore, EstGUZ753 showed marked tolerance in methanol. The activity of EstGUZ753 increased up to 1.16-fold in 90% methanol after 72 h, and the half-life in it was 336 h. The crude fermentation broth of EstGUZ753 was immobilized directly onto the epoxy resin (Lx-105s), and the immobilized EstGUZ753 exhibited a 99% conversion for cinnamyl acetate synthesis at a high cinnamyl alcohol concentration in 6 h (cinnamyl alcohol: 1.0 M, enzyme dosage: 3%). These characteristics of EstGUZ753 indicated its great potential for organic synthesis. A novel esterase gene from Geobacillus uzenensis DSMZ 13551 was first cloned and expressed in Pichia pastoris KM71. Esterase EstGUZ753 showed marked thermostability at high temperature and tolerance in methanol. Furthermore, the crude fermentation broth of EstGUZ753 was immobilized directly onto the epoxy resin, and immobilized EstGUZ753 exhibited a 99% conversion for cinnamyl acetate synthesis.

Efficient production of \u03b1-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate

BackgroundDiacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl.ResultsBy expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate.ConclusionsAn efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.

Novel Synthesis Strategy for Biocatalyst: Fast Purification and Immobilization of His- and ELP-Tagged Enzyme from Fermentation Broth.

Inspired by natural biomineralization process, inorganic phosphates system has been selected as a candidate for the encapsulation of enzyme; however, during the long-term fabrication process, the loss of enzyme activity is unavoidable, and the biomimetic mineralization mechanism is still poorly understood. Meanwhile, the purification process plays a key role in the preparation of immobilized enzyme with high enzyme loading and activity, while the rapid, low-cost, and eco-friendly purification of biocatalyst from crude fermentation broth remains a critical challenge in biochemical engineering. Here, a binary tag composed of elastin-like polypeptide (ELP) and His-tag was presented for the first time to be fused with β-glucosidase (Glu) to construct a recombinant Glu-linker-ELP-His (GLEH) with the aim of developing a fast synthesis strategy combining purification and immobilization processes for a biocatalyst with better stability and recyclability. The purification fold and activity recovery of GLEH reached 18.1 and 95.2%, respectively, once a single inverse transition cycling was conducted at 25 °C for 10 min. Then, efficient biomineralization of hybrid enzyme-Cu3(PO4)2 nanoflowers was realized in 15 min by the action of His-tag and ultrasonic-assisted reaction method. The activity recovery and relative activity reached the maximum at 90.3 and 111.0%, respectively. We demonstrate that the crystal growth process of a hybrid nanoflower involves obvious nucleation, self-assembly, and the Ostwald ripening process, and the enzyme GLEH acts as a "binder" to assemble Cu3(PO4)2 nanoflakes. The immobilized GLEH nanoflowers show outstanding operation stability and recyclability, and their catalytic efficiency is close to that of free Glu.

Downstream separation and purification of succinic acid from fermentation broths using spent sulphite liquor as feedstock

Spent sulphite liquor produced as side stream from sulphite pulping of Eucalyptus globulus hardwood could be used for the separation of lignosulphonates by nanofiltration in the retentate stream and succinic acid production via fermentation of the permeate stream by Actinobacillus succinogenes or Basfia succiniciproducens. The potential integration of this process in conventional pulp mills towards the development of a novel biorefinery is dependent on the efficient downstream separation of succinic acid crystals at high yield and purity. This study focuses on the evaluation of five downstream separation processes, namely calcium precipitation, direct crystallisation using acidification or cation-exchange resins, salting-out and reactive extraction, for the purification of succinic acid from crude fermentation broths. Reactive extraction using trioctylamine in 1-hexanol and direct crystallisation coupled with cation-exchange resins led to succinic acid recovery yields of 73% and 79%, respectively. 1H NMR analysis showed that these downstream separation processes led to succinic acid crystal purities of ca 98.5% for reactive extraction and higher than 99% for the direct crystallisation method coupled with cation-exchange resins with no detectable acetic acid content when re-crystallisation was employed. It has been demonstrated that succinic acid produced via fermentation using side streams from pulp and paper mills could be separated at high purity and yield from crude fermentation broths rendering feasible its utilisation for poly(butylene succinate) production.

Chromatographic separation of phenyllactic acid from crude broth using cryogels with dual functional groups

Phenyllactic acid (PLA) is an important organic acid with wide antimicrobial activities against gram-positive and gram-negative bacteria and some fungi. This interesting compound can be synthesized by the microbial fermentation or the bioconversion using phenylpyruvic acid (PPA) as the key substrate and microorganisms as the whole-cell biocatalysts. However, the isolation of high-purity PLA with a high recovery from the crude fermentation or conversion broth is a challenging task. In this work, the separation of PLA from the crude conversion broth prepared by employing Lactobacillus buchneri cells as the whole-cell catalysts was achieved by the chromatography using the poly(hydroxyethyl methacrylate) (pHEMA)-based cryogel with a combination of anion-exchange and hydrophobic benzyl groups. The static adsorption behaviors of PLA under different salt concentrations and the adsorption capacities of PLA on the cryogel were measured experimentally. The chromatographic performance of PLA from the crude conversion broth was compared with that from the clarified broth. The results showed that the pHEMA-based cryogel has a high capacity of PLA, i.e., 14.64 mg mL−1 cryogel, and the adsorption of PLA was influenced by the salt concentration. By using deionized water as running buffer, PLA with a high purity of 97.6% was obtained with one step elution using 0.3 M NaCl as the elution solution with the recovery at the range of 80.2–90.8% from crude feedstock without any pretreatment at various flow velocities. These values were close to those obtained for the clarified broth, i.e., the purity of 98.4% and the recovery of 92.3% under the same chromatography conditions at 1 cm min−1. The cryogel was then applied to separate PLA from clarified feedstock, high purity (>96.7%) and recovery (>91.4%) of PLA were found with 20 cycles, which verified the selectivity and robustness of prepared pHEMA-VBTAC cryogel. Therefore, the chromatography using pHEMA-based cryogel with the dual functional groups is an effective approach for the isolation of PLA directly from the crude bioconversion broth and thus could be interesting in the separation and production of high-purity PLA in industry.

Techniques for the recovery of volatile aroma compounds from biochemical broth: A review

AbstractFlavours, fragrances, or aromas are valuable chemicals, with a broad range of industrial applications. The consumer's preference for ‘naturally’ manufactured aromas drives the development of biochemical production, which requires suitable product recovery. In this study, different industrially established recovery techniques, their advantages and limitations, and their possible application for aroma recovery from crude fermentation broth are reviewed. Among several techniques discussed, absorption, adsorption, and pervaporation appear to be particularly suitable for the recovery of aromas from highly diluted biochemical mixtures.

Recovery and primary purification of bacteriophage M13 using aqueous two‐phase systems

AbstractBACKGROUNDBacteriophage M13 is an Escherichia coli‐specific non‐lytic filamentous virus commonly used in applications ranging from antibody screening and nanomaterial construction to drug delivery, among others. In this tenor, alternative methods for the fractionation, recovery and partial purification of phage particles are desired. In this work, the use of aqueous two‐phase systems (ATPS) was evaluated as an alternative method for the recovery of phage particles.RESULTSThe partition behavior of M13 in PEG–salt and ionic liquid (IL)–salt ATPS was characterized using a pre‐purified feedstock. In PEG‐salt ATPS, M13 was preferentially partitioned to the interface. In IL ATPS, however, M13 showed a high‐top phase preference with recovery yields above 65%. Selected systems were tested for the extraction of M13 from a crude fermentation broth. From crude broth, a PEG 400‐potassium phosphate system with volume ratio (VR) of 1 and 25% w/w tie line length (TLL) gave the best M13 top phase recovery (83%) and purification fold (18.2) in terms of total protein concentration.CONCLUSIONSThe results presented here demonstrate the practical application of ATPS as an efficient process for the primary recovery and partial purification of M13 and represent the first study of the extraction of viral particles directly from a crude broth as well as the use of IL‐Salt ATPS. © 2017 Society of Chemical Industry

Aquatic micro-pollutants removal with a biocatalytic membrane prepared by metal chelating affinity membrane chromatography

Biocatalytic membranes are promising to remove micro-pollutants in aqueous environment due to their mild and green operation condition. However, more efforts need to be devoted to improving their removal efficiency and stability. In this study, metal chelating affinity membrane chromatography (MCAMC) was used to construct a biocatalytic membrane by selectively capturing laccase from a crude fermentation broth. Metal ions had a significant effect on the activity of the immobilized laccase and copper ion was the best choice. A pH of 4.5 was selected for laccase adsorption and its loading seemed the same under flow rates from 0.5 to 10mLmin−1 thanks to the inherent convective transport of membrane chromatography. The pH value and salt concentration in the storage buffer had an obvious effect on the stability of the immobilized laccase, and the prepared biocatalytic membrane retained 87% of initial activity after 20days storage. When applying such membrane to micro-pollutant removal (taking bisphenol A (BPA) as an example), a high BPA removal efficiency (99.3%) could be obtained. The biocatalytic membranes could be operated at a high flux of 50Lm−2h−1 without recycling the permeate into the feed, and its throughput and BPA removal rate were superior to the most results in the literature. However, BPA removal decline (from 99.6% to 56.6% after five cycles) occurred during the successive water treatment due to the membrane fouling caused by BPA polymerization products. Membrane regeneration could be achieved by simple elution-cleaning-reloading, and the laccase activity and BPA removal were fully recovered.

Membrane chromatography for fast enzyme purification, immobilization and catalysis: A renewable biocatalytic membrane

Enzyme purification and subsequent immobilization usually require multiple steps and consume plenty of chemicals. Traditional dipping incubation operation is lengthy and the interior of porous carrier is not fully exploited due to the diffusion barrier. To solve these problems, one-step enzyme purification and immobilization was developed based on membrane chromatography concept. A membrane adsorber was prepared by polydopamine coating on a PVDF microfiltration membrane (or on a cheap and disposable cellulose filter paper) and subsequent polyethyleneimine grafting, without any solvent usage. Laccase was selectively immobilized on such membrane adsorber (enzyme activity: 39.9±4.4U/mL; expressed activity: 19.6±3.0%) by directly capturing it from a crude fermentation broth without pH adjustment under flow-through mode. The immobilized laccase had a high purity of 92.2%, while the membrane permeability decreased due to the adsorption of laccase and impurities (“membrane fouling”). Thanks to the dominant convective transport, the enzyme loading was high and much faster than that by dipping incubation (5min vs. 80min). Moreover, enzyme reloading on membrane adsorber was easily realized by reversible desorption-adsorption process. The constructed biocatalytic membrane exhibited a commendable reusability for bisphenol A (BPA) removal only driven by its gravity.

Nonionic surfactants induced cloud point extraction of Polyhydroxyalkanoate from Cupriavidus necator

ABSTRACTPolyhydroxyalkanoate synthesized by Cupriavidus necator DSM 428 was purified from the crude fermentation broth as such by performing nonionic surfactants (Triton X100, Triton X114 & Tergitol 6) induced cloud point extraction. Polyhydroxyalkanoate was extracted into the micelle-rich bottom phase (coacervate phase), while most of the cellular impurities partitioned into the aqueous phase. Cloud point temperatures and the extraction efficiency of different cloud point systems were studied at different pH value and in the presence of additives. Maximum extraction of biopolymer was achieved (recovery of 84.4%) with a purity of 92.49% at 3 pH with the addition of 0.1 M ammonium chloride in the mixed surfactant system at a reduced cloud point temperature of 33°C.

Electrocatalytic upgrading of itaconic acid to methylsuccinic acid using fermentation broth as a substrate solution

An electrocatalytic transformation of biomass based platform chemicals is feasible even using crude fermentation broth.

Towards the upgrading of fermentation broths to advanced biofuels: a water tolerant catalyst for the conversion of ethanol to isobutanol

The conversion of methanol/ethanol mixtures to isobutanol with the pre-catalyst trans-[RuCl2(dppm)2] (1) is tolerant to the addition of water to the system, achieving an isobutanol yield of 36% at 78% selectivity with water concentrations typical of that of a crude fermentation broth.

Aqueous two phase purification of xylanase obtained from a bacterial isolate

The aim of this work was to extract and to purify xylanase produced by the bacillus strain from the crude fermentation broth using aqueous two phase system (ATPS).Xylanase is an important industrial enzyme that finds use in several industries like paper and pulp, textiles, food and feed and production of bioethanol from agri-residues. There is a huge demand for a cost effective xylanase in the industry. The xylanase was extracted by partitioning in ATPS composed of phosphate and polyethylene glycol (PEG). The technique resulted in 1.35 fold purification. The specific activity of the xylanase obtained was 6.27mu/mg

Antimicrobial activity of Streptomyces spp. isolates from vegetable plantation soil

Fifteen Streptomyces isolates were isolated from soil in some different location on vegetable plantation at agriculture standard condition. The isolates were assessed for their antibacterial activity against Mycobacterium tuberculosis (MTB) ATCC H37RV and mycobacterial which isolated from Dr. Soetomo Hospital patients in Surabaya. The Interna-tional Streptomyces Project 4 (ISP4) and Middlebrook 7H9 (MB7H9) wwere used as growth or fermentation medium. The screening of inhibition activity was performed using turbidimetry and spot-test on agar medium. Results shown that 33.3% of the isolates (5 isolates) have anti-mycobacterial activities. The first line anti tuberculosis drug rifampicin, (RIF), ethambutol (EMB), isoniazid (INH), and pyrazinamide (PZA) were used as standards or positive controls with concentration 20 ppm. Optical density of crude fermentation broth concentrated from five isolates relatively lower than five anti-tuberculosis drug activity standard, although their activities against some microbial were similar to the stand-ard at spot-test. The most efficient isolate shown anti-mycobacterial activity was Streptomyces B10 which identified as Streptomyces violaceousniger. In addition, fatty acid methyl ester (FAME) profile of gas chromatography-mass spec-trometry chromatogram of each isolates were studied and compared to Streptomyces spp.