Rising greenhouse gas concentrations in the atmosphere are having deleterious effects on biotic and abiotic systems, causing serious global concerns. Bioprocess technology is able to utilize these gases to manufacture high-value products and provide an excellent alternative to currently expensive conventional carbon sources in use. Methanotrophs are ubiquitous and utilize reduced carbon substrates without C-C bonds, such as methane, methanol, methylamine, and formaldehyde. Notably, methanotrophs produce single-cell proteins, polyhydroxyalkanoates, methanol, and exopolysaccharides, using a unique metabolic pathway during their growth process. Many industries have developed copyright-protected bioprocesses to utilize methane through methanotrophic pathways and manufacture high-value products. Among methanotrophs, Methylosinus trichosporium OB3b is one of the most important methane oxidizing bacteria; it has been studied extensively to identify potential applications including methanol and biopolymer production, and the bioremediation of environmental contaminants. This paper summarizes the characteristics and versatile role of methanotrophs and Methylosinus trichosporium OB3b. In addition, commercially established biological conversions of methane were constituted.

Abstract: The α-amylase is one of the most promising commercial enzymes with tremendous applications in various industries. Microbial α-amylase shares almost 25-30% of the enzyme market due to its catalytic function in several industries, including sugar, detergent, paper, textile, pharmaceutical industries, etc. The α-amylase hydrolyzes glycosidic linkages of structural components of starch, resulting in maltose, glucose, and high fructose syrups. Starch, the second most abundant organic substance on the Earth, is a readily available, low-cost renewable substrate mainly used in biorefinery and food industries. Amylases are ubiquitous in nature due to their involvement in carbohydrate metabolism. The α-amylases of microbial origin have technical advantages as compared to animal and plant origin. Considering physicochemical properties, bacterial α-amylases are most diverse. However, for industrial purposes, these properties of the biocatalyst, either individually or in a combination, are required to modify through genetic and protein engineering according to the targeted process. The review presents an overview of the current findings of microbial sourced α- amylases, commercial applications, market trends in relevant industries, and achieved improvements in thermostability, catalytic function, pH tolerance, substrate, and product specificities through recombinant DNA technology and protein engineering.

Harmful algal blooms (HABs) is a rapid increase or accumulation in the population of harmful microalgae in freshwater and marine aquatic system, and can harm the health of the environment, plants or animals.Conventional chemical and physical techniques to mitigate HABs such as chlorination, ozonation, ultrasonic treatment, and clay-based flocculation are limited by the low efficiency and risk of destroying the aquatic ecosystems.Therefore, host-specific virus-like particles (VLPs) as an alternative biological method for the efficient control of harmful microalgae that can be combined with chemically synthesized algicidal compound has been successfully performed in this lab.This system was efficient for algicidal activity but also involved several significant disadvantages such as difficult purification and less production rate.To overcome these issues, we attempted to develop a low-cost, constitutive overexpression, and easy purification system of viral capsid protein to obtain large amounts VLPs.The capsid protein of HcRNAV34 as a singlestranded RNA virus that infects the toxic dinoflagellate, Het-erocapsa circularisquama, was successfully expressed in E. coil without inducer.The maximal condition for production of HcRNAV34 capsid protein in the E. coil BL21(DE3) was optimized, and then finally 3.25 g/ℓ (wet weight) cell mass was obtained from 30 L reactor containing M9 medium at 37 o C for 20 h.The expressed insoluble capsid protein was partially purified by low speed centrifugation and labelled with green fluorescent FITC.The resulting protein was visualized on the cell surface of H. circularisquama 9433 and 92-1, the hosts of native HcRNAV34 virus.In addition, HcRNAV34 VLPs absorbed with TD49 as algicidal substance showed a high algicidal effect against H. circularisquama 9433 and 92-1 in a host-specific manner.This host target-specific VLPs can be used for the management of HABs in aquatic ecosystems.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

In this study, we investigated the potential of Paenibacillus kribbensis CU01 in producing fusaricidin, a strong antifungal substance, via optimization of metal ions and carbon and nitrogen source, and continuous fermentation. In the cultivation of a 2-l batch, maximal production of fusaricidins (581 mg l −1 ) was achieved in a modified M9 medium containing metal ions, 10 g l −1 glucose, and 1 g l −1 ammonium chloride. Most of glucose was consumed at a rate of 0.74 g l −1 h −1 within 24 h and fusaricidin production began 15 h after batch cultivation. Continuous fermentation was performed using a 7-l fermenter with 2-l working volume of modified M9 medium containing 10 g l −1 glucose, 1 × 10 −3 M FeSO 4 , and 1 × 10 −6 M MnCl 2 . After 24 h of the start of cultivation, fresh M9 medium was continuously supplied at a flow rate of 2.5 ml min −1 , and simultaneously, the same amount of cell culture broth was removed. In a continuous system, the highest fusaricidin concentration (579 mg l −1 ) was obtained using a dilution rate of 0.075 h −1 with an average productivity of 10.4 mg l −1 h −1 for 24 to 72 h of incubation. Based on these results, it was found that fusaricidin production using P. kribbens CU01 strain increased by at least 28 times the values reported in previous studies.

Methylomonas koyamae LM6 is a potential methanotrophic bacterium of interest for methane bioconversion. Here, we report the complete genome sequence of M. koyamae LM6, which contains 4,337 predicted open reading frames on one chromosome (4,894,002 bp) and one plasmid (186,658 bp), with genes involved in methane oxidation.

With the progress of biotechnological research and improvements made in bioprocessing with pure cultures, microbial consortia have gained recognition for accomplishing biological processes with improved effectiveness. Microbes are indispensable tool in developing bioprocesses for the production of bioenergy and biochemicals while utilizing renewable resources due to technical, economic and environmental advantages. They communicate with specific cohorts in close proximity to promote metabolic cooperation. Use of positive microbial associations has been recognized widely, especially in food industries and bioremediation of toxic compounds and waste materials. Role of microbial associations in developing sustainable energy sources and substitutes for conventional fuels is highly promising with many commercial prospects. Detoxification of chemical contaminants sourced from domestic, agricultural and industrial wastes has also been achieved through microbial catalysis in pure and co-culture systems. Methanotrophs, the sole biological sink of greenhouse gas methane, catalyze the methane monooxygenasemediated oxidation of methane to methanol, a high energy density liquid and key platform chemical to produce commodity chemical compounds and their derivatives. Constructed microbial consortia have positive effects, such as improved biomass, biocatalytic potential, stability etc. In a methanotroph-heterotroph consortium, non-methanotrophs provide key nutrient factors and alleviate the toxicity from the culture. Non-methanotrophic organisms biologically stimulate the growth and activity of methanotrophs via production of growth stimulators. However, methanotrophs in association with co-cultured microorganisms are in need of further exploration and thorough investigation to study their interaction mode and application with improved effectiveness.

Phaeodactylum tricornutum is an emerging diatom in the microalgal biotechnology field for biofuel application. P. tricornutum B2089 was incubated for 13 days in a 3 L flask containing f/2 medium mixed with oceanic sediment extracts. To select the most effective pretreatment process (cell destruction method) of P. tricornutum B2089 for lipid extraction, various mechanical methods including autoclave, bead beating, sonication, French-press, and microwave were tested. Of these, enhanced lipid extraction was obtained through microwave treatment (1200W for 20 minutes at 2450 ㎒, 150℃). Under this condition, the lipid content from P. tricornutum B2089 was 53.3% (w/w) of dry cell weight or 3.4 g/L and the lipid contained palmitoleic acid (C16:1) and oleic acid (C18: 1) as major fatty acids.

This study demonstrates the effects of growth stimulators in oceanic sediment on biomass and lipid production of two oleaginous microalgae, Botryococcus braunii LB572 and Phaeodactylum tricornutum B2089. At the optimal mixing ratio of culture medium and oceanic sediment extract of 6:4 (v/v), specific growth rates of B. braunii LB572 and P. tricornutum B2089 increased 13.0- and 11.3-fold, respectively, compared to a sediment-free medium. The maximum biomass and lipid productions of B. braunii LB572 were 5.54 and 3.09 g L−1, and those of P. tricornutum B2089 were 6.41 and 3.61 g L−1, respectively, indicating that biomass and lipid production in both microalgae increased at least 6- and 8-fold, respectively. Thus, their cultivation time was reduced by at least 6 days. A positive effect of nitrate in the sediment on biomass and lipid production was not found. Fe3+ and Ca2+ promoted biomass and lipid production as their concentrations increased. However, metal ion concentration is not as critical to biomass and lipid production as humic acid, a chelating substance to enhance bioavailability of metal ions to the microalgae. The optimal humic acid concentration for maximal biomass and lipid production was 80 mg L−1, which is the concentration contained in the culture medium mixed with sediment extract at a ratio of 6:4 (v/v). Thus, low-cost oceanic sediment can supply sufficient growth stimulators, especially humic acid, for mass production of biomass and lipid in both microalgae.

Several methods including microwave, Frenchpress, autoclave, bead-beating, ultrasonication, and osmotic shock were compared to identify the most effective microalgal cell disruption method. Botryococcus braunii LB572 was cultured in 5 L flasks containing JM medium mixed with oceanic sediment extract for 13 days. Among the methods tested, enhanced lipid extraction was achieved through microwave treatment (2450MHz, 1250W at 150°C for 20 min). Oleic (C18:1), linolenic (C18:3), and palmitic acids (C16:0) were found to be the major fatty acids among the C14-C24 acids from extracted lipid. In addition, the optimal conditions of transesterification were as follows: 70 mL of methanol, 6 mL of sulfuric acid, 8 mL of chloroform, and boiling at 100°C for 30 min; 85.4% of C14-C24 FAME and 78.5% of C16-C18 FAME were esterified from transesterifiable lipids.

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MPT (MDHMas), was determined at 1.7 Å resolution. The active form of MDHMas (or MDHIMas) is a heterotetrameric α2β2, where each β-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two β-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a β-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg2+ ion, instead of the Ca2+ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the β-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHIMas integrity in the sea water environment to provide a firm basis for complex formation with MxaJMas or Cyt cL. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.

The growth properties of Panax ginseng hairy roots transformed by Agrobacterium rhizogenes were compared between flask and aerated column or stirred bioreactor. In flask cultures, sucrose, initially 30 g/L, was nearly exhausted after 45 d of culture. The pH of the medium dropped from 5.5 to 4.96 after 10 d, but afterward it gradually increased to 6.4. After 45 d, hairy roots grew about 16-folds. The growth rate of hairy roots in air-bubble column or stirred bioreactor cultures was 1.13 (1.11) to 1.23 (1.20) g fresh wt (dry wt)/(g of cells·d), respectively. For both bioreactors, growth was about three times as high as in the flask cultivation.

We examined the effects of the algicide thiazolidinedione (TD49) and yellow clay on Chattonella marina and assessed their ecological risk for the entire planktonic community. Mesocosm (1000 L) exposure experiments were employed to investigate time-course responses over 9 days. The growth of C. marina was controlled at ≥0.4 μM TD49 but not inhibited in yellow clay treatments. Although the algicidal activity of the 0.4-μM TD49 + 0.4 kg t−1 yellow clay treatment for C. marina was high (72.5 % at 24 h), target alga regrowth occurred. In all treatments, inorganic nutrients such as nitrate + nitrite and phosphate decreased following commencement of the experiment but were >1 μM (limitation concentration) at days 5 and 6, even though consumption pattern of those nutrients was influenced by the TD49 concentration. Depletion of silicate in initial stages played an important role in controlling the shift from diatoms including Chaetoceros and Skeletonema spp. to cryptophytes. Zooplankton were not affected by even the highest the yellow clay treatments and TD concentration of <0.8 μM, but their abundance significantly reduced after day 1 at 0.8 μM TD49. Zooplankton nauplii gradually increased to the end of the experimental period, implying that TD49 may have a limited effect on zooplankton communities. The initial dosing concentration of each substance and the fate of nutrients following algicide application were critical in determining the timing of shifts in the phytoplankton and zooplankton species composition, as well as the algicidal effect on the target alga.

ABSTRACTMxaJ is a component of type II methanol dehydrogenase (MDH) that mediates electron transfer during methanol oxidation in methanotrophic bacteria. However, little is known about how MxaJ structurally cooperates with MDH and Cytochrome cL. Here, we report for the first time the crystal structure of MxaJ. MxaJ consists of eight α‐helices and six β‐strands, and resembles the “bi‐lobate” folding architecture found in periplasmic binding proteins. Distinctive features of MxaJ include prominent loops and a β‐strand around the hinge region supporting the ligand‐binding cavity, which might provide a more favorable framework for interacting with proteins rather than small molecules. Proteins 2017; 85:1379–1386. © 2017 Wiley Periodicals, Inc.

A bacterial strain showing strong antifungal activity was isolated from yellow loess and was identified as Paenibacillus kribbensis CU01. Insoluble mucoidal polymers were separated from M9 culture medium via low-speed centrifugation. Most antifungal activity was associated with substances in the insoluble precipitate, which was purified by reverse phase high performance liquid chromatography. Purified fractions were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Two major ion peaks with mass-to-charge ratio values (m/z) at 883.6 and 897.6 were revealed. After alkaline hydrolysis and sequence analysis, two cyclic depsipeptides were identified as, fusaricidin A and fusaricidin B. Their production was significantly increased by the addition of glucose, Fe2+, and Mn2+ to M9 medium. Maximum concentrations of produced fusaricidin A and fusaricidin B at flask-scale comprised 460 mg L-1 and 118 mg L-1, respectively: the highest production concentrations yet reported in the literature. This demonstrates that P. kribbensis CU01 has enormous commercial potential for the mass production of fusaricidin.

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m2/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16–C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.