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Applied Microbiology and Biotechnology (v.96, #3)

Conversion of CO₂ into biomass by microalgae: how realistic a contribution may it be to significant CO₂ removal? by F. Gabriel Acién Fernández; C. V. González-López; J. M. Fernández Sevilla; E. Molina Grima (pp. 577-586).

Microalgae have been proposed as a CO₂ removal option to contribute to climate change avoidance and problems coming from the use of fossil fuels. However, even though microalgae can be used to fix CO₂ from air or flue gases, they do not permit long-term CO₂ storage because they are easily decomposed. On the other hand, microalgae can contribute to an enhancement in human sustainability by producing biofuels as an alternative to fossil fuels in addition to the production of other useful chemicals and commodities. Moreover, microalgae can contribute to enhancing the sustainability of waste treatment processes, reducing the energy consumed, and improving the recycling of nutrients contained within them. This paper reviews the potential contribution of these processes and the existing knowledge in these areas.

Keywords: CO₂ fixation; Microalgae; Biofuels; Wastewater; Biogas

How spectroscopy and microspectroscopy of degraded wood contribute to understand fungal wood decay by Karin Fackler; Manfred Schwanninger (pp. 587-599).

Nuclear magnetic resonance, mid and near infrared, and ultra violet (UV) spectra of wood contain information on its chemistry and composition. When solid wood samples are analysed, information on the molecular structure of the lignocellulose complex of wood e.g. crystallinity of polysaccharides and the orientation of the polymers in wood cell walls can also be gained. UV and infrared spectroscopy allow also for spatially resolved spectroscopy, and state-of-the-art mapping and imaging systems have been able to provide local information on wood chemistry and structure at the level of wood cells (with IR) or cell wall layers (with UV). During the last decades, these methods have also proven useful to follow alterations of the composition, chemistry and physics of the substrate wood after fungi had grown on it as well as changes of the interactions between the wood polymers within the lignocellulose complex caused by decay fungi. This review provides an overview on how molecular spectroscopic methods could contribute to understand these degradation processes and were able to characterise and localise fungal wood decay in its various stages starting from the incipient and early ones even if the major share of research focussed on advanced decay. Practical issues such as requirements in terms of sample preparation and sample form and present examples of optimised data analysis will also be addressed to be able to detect and characterise the generally highly variable microbial degradation processes within their highly variable substrate wood.

Keywords: White-rot fungi; Brown-rot fungi; Wood degradation; Nuclear magnetic resonance spectroscopy; NMR spectroscopy; Near infrared spectroscopy; Mid infrared spectroscopy; NIR spectroscopy; MIR spectroscopy; UV spectroscopy; Microspectroscopy; Imaging

Flavour-active wine yeasts by Antonio G. Cordente; Christopher D. Curtin; Cristian Varela; Isak S. Pretorius (pp. 601-618).

The flavour of fermented beverages such as beer, cider, saké and wine owe much to the primary fermentation yeast used in their production, Saccharomyces cerevisiae. Where once the role of yeast in fermented beverage flavour was thought to be limited to a small number of volatile esters and higher alcohols, the discovery that wine yeast release highly potent sulfur compounds from non-volatile precursors found in grapes has driven researchers to look more closely at how choice of yeast can influence wine style. This review explores recent progress towards understanding the range of ‘flavour phenotypes’ that wine yeast exhibit, and how this knowledge has been used to develop novel flavour-active yeasts. In addition, emerging opportunities to augment these phenotypes by engineering yeast to produce so-called grape varietal compounds, such as monoterpenoids, will be discussed.

Keywords: Aroma; Flavour; Fermented beverages; Wine; Yeast

Sweet-taste-suppressing compounds: current knowledge and perspectives of application by Maud Sigoillot; Anne Brockhoff; Wolfgang Meyerhof; Loïc Briand (pp. 619-630).

Sweet-tasting compounds are recognized by a heterodimeric receptor composed of the taste receptor, type 1, members 2 (T1R2) and 3 (T1R3) located in the mouth. This receptor is also expressed in the gut where it is involved in intestinal absorption, metabolic regulation, and glucose homeostasis. These metabolic functions make the sweet taste receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions such as diabetes. Existing sweet taste inhibitors or blockers that are still in development would constitute promising therapeutic agents. In this review, we will summarize the current knowledge of sweet taste inhibitors, including a sweet-taste-suppressing protein named gurmarin, which is only active on rodent sweet taste receptors but not on that of humans. In addition, their potential applications as therapeutic tools are discussed.

Keywords: Gurmarin; Taste receptor; Inhibitor; Sweetener; GPCR; Gymnema sylvestre

Microalgal carbohydrates: an overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels by Giorgos Markou; Irini Angelidaki; Dimitris Georgakakis (pp. 631-645).

Microalgal biomass seems to be a promising feedstock for biofuel generation. Microalgae have relative high photosynthetic efficiencies, high growth rates, and some species can thrive in brackish water or seawater and wastewater from the food- and agro-industrial sector. Today, the main interest in research is the cultivation of microalgae for lipids production to generate biodiesel. However, there are several other biological or thermochemical conversion technologies, in which microalgal biomass could be used as substrate. However, the high protein content or the low carbohydrate content of the majority of the microalgal species might be a constraint for their possible use in these technologies. Moreover, in the majority of biomass conversion technologies, carbohydrates are the main substrate for production of biofuels. Nevertheless, microalgae biomass composition could be manipulated by several cultivation techniques, such as nutrient starvation or other stressed environmental conditions, which cause the microalgae to accumulate carbohydrates. This paper attempts to give a general overview of techniques that can be used for increasing the microalgal biomass carbohydrate content. In addition, biomass conversion technologies, related to the conversion of carbohydrates into biofuels are discussed.

Keywords: Biofuels; Biomass conversion technologies; Carbohydrates; Microalgae; Nutrient starvation

Development of micro-shock wave assisted dry particle and fluid jet delivery system by S. G. Rakesh; Divya Prakash Gnanadhas; Uday Sankar Allam; Karaba N. Nataraja; P. K. Barhai; Gopalan Jagadeesh; Dipshikha Chakravortty (pp. 647-662).

Small quantity of energetic material coated on the inner wall of a polymer tube is proposed as a new method to generate micro-shock waves in the laboratory. These micro-shock waves have been harnessed to develop a novel method of delivering dry particle and liquid jet into the target. We have generated micro-shock waves with the help of reactive explosive compound [high melting explosive (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and traces of aluminium] coated polymer tube, utilising ∼9 J of energy. The detonation process is initiated electrically from one end of the tube, while the micro-shock wave followed by the products of detonation escape from the open end of the polymer tube. The energy available at the open end of the polymer tube is used to accelerate tungsten micro-particles coated on the other side of the diaphragm or force a liquid jet out of a small cavity filled with the liquid. The micro-particles deposited on a thin metal diaphragm (typically 100-μm thick) were accelerated to high velocity using micro-shock waves to penetrate the target. Tungsten particles of 0.7 μm diameter have been successfully delivered into agarose gel targets of various strengths (0.6–1.0 %). The device has been tested by delivering micro-particles into potato tuber and Arachis hypogaea Linnaeus (ground nut) stem tissue. Along similar lines, liquid jets of diameter ∼200–250 μm (methylene blue, water and oils) have been successfully delivered into agarose gel targets of various strengths. Successful vaccination against murine salmonellosis was demonstrated as a biological application of this device. The penetration depths achieved in the experimental targets are very encouraging to develop a future device for biological and biomedical applications.

Keywords: Shock wave; Micro-shock wave; Liquid jet delivery system; Needle-less vaccination; Dry particle delivery system

The effect of iron-chelating agents on Magnetospirillum magneticum strain AMB-1: stimulated growth and magnetosome production and improved magnetosome heating properties by Edouard Alphandéry; Matthieu Amor; François Guyot; Imène Chebbi (pp. 663-670).

The introduction of various iron-chelating agents to the Magnetospirillum magneticum strain AMB-1 bacterial growth medium stimulated the growth of M. magneticum strain AMB-1 magnetotactic bacteria and enhanced the production of magnetosomes. After 7 days of growth, the number of bacteria and the production of magnetosomes were increased in the presence of iron-chelating agents by factors of up to ∼2 and ∼6, respectively. The presence of iron-chelating agents also produced an increase in magnetosome size and chain length and yielded improved magnetosome heating properties. The specific absorption rate of suspensions of magnetosome chains isolated from M. magneticum strain AMB-1 magnetotactic bacteria, measured under the application of an alternating magnetic field of average field strength ∼20 mT and frequency 198 kHz, increased from ∼222 W/g_Fe in the absence of iron-chelating agent up to ∼444 W/g_Fe in the presence of 4 μM rhodamine B and to ∼723 W/g_Fe in the presence of 4 μM EDTA. These observations were made at an iron concentration of 20 μM and iron-chelating agent concentrations below 40 μM.

Keywords: Magnetosomes; Magnetotactic bacteria; Iron-chelating agents; Siderophore; Magnetic hyperthermia; Alternating magnetic field

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19 by Ju Hyun Park; Zesong Wang; Hee-Jin Jeong; Hee Ho Park; Byung-Gee Kim; Wen-Song Tan; Shin Sik Choi; Tai Hyun Park (pp. 671-683).

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

Keywords: CHO cells; rHuEPO; 30Kc19 protein; Productivity; Glycosylation

Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron limitation by Christian Löser; Thanet Urit; Sylvia Förster; Anton Stukert; Thomas Bley (pp. 685-696).

The ability of Kluyveromyces marxianus to convert lactose into ethyl acetate offers a chance for an economic reuse of whey. Former experiments with K. marxianus DSM 5422 proved limitation of growth by iron (Fe) or copper as a precondition for significant ester synthesis. Several aerobic batch and chemostat cultivations were done with whey-borne media of a variable Fe content for exploring the effect of Fe on growth, the Fe content of biomass, and metabolite synthesis. At low Fe doses, Fe was the growth-limiting factor, the available Fe was completely absorbed by the yeasts, and the biomass formation linearly depended on the Fe dose governed by a minimum Fe content in the yeasts, x _Fe,min. At batch conditions, x _Fe,min was 8.8 μg/g, while during chemostat cultivation at D = 0.15 h⁻¹, it was 23 μg/g. At high Fe doses, sugar was the growth-limiting factor, Fe was more or less absorbed, and the formed biomass became constant. Significant amounts of ethyl acetate were only formed at Fe limitation while high Fe doses suppressed ester formation. Analysis of formed metabolites such as glycerol, pyruvate, acetate, ethanol, ethyl acetate, isocitrate, 2-oxoglutarate, succinate, and malate during chemostat cultivation allowed some interpretation of the Fe-dependent mechanism of ester synthesis; formation of ethyl acetate from acetyl-SCoA and ethanol is obviously initiated by a diminished metabolic flux of acetyl-SCoA into the citrate cycle and by a limited oxidation of NADH in the respiratory chain since Fe is required for the function of aconitase, succinate dehydrogenase, and the electron-transferring proteins.

Keywords: Kluyveromyces marxianus ; Whey; Ethyl acetate; Chemostat; Iron limitation; Metabolites

Characterization of alcohol dehydrogenase 3 of the thermotolerant methylotrophic yeast Hansenula polymorpha by Surisa Suwannarangsee; Seonghun Kim; Oh-Cheol Kim; Doo-Byoung Oh; Jeong-Woo Seo; Chul Ho Kim; Sang Ki Rhee; Hyun Ah Kang; Warawut Chulalaksananukul; Ohsuk Kwon (pp. 697-709).

In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha. However, unlike the cytosolic HpADH1, HpADH3 appears to be a mitochondrial enzyme, as a mitochondrial targeting extension exists at its N terminus. The recombinant HpADH3 overexpressed in Escherichia coli showed similar catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The HpADH3 displayed substrate specificities with clear preferences for medium chain length primary alcohols and acetaldehyde for an oxidation reaction and a reduction reaction, respectively. Although the H. polymorpha ADH3 gene was induced by ethanol in the culture medium, both an ADH isozyme pattern analysis and an ADH activity assay indicated that HpADH3 is not the major ADH in H. polymorpha DL-1. Moreover, HpADH3 deletion did not affect the cell growth on different carbon sources. However, when the HpADH3 mutant was complemented by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium. In contrast, in a xylose medium, the ethanol production was dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain. Taken together, our results suggest that the expression of HpADH3 would be an ideal engineering target to develop H. polymorpha as a substrate specific bioethanol production strain.

Keywords: Alcohol dehydrogenase; ADH3; Hansenula polymorpha ; Ethanol production; Xylose fermentation

Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2 by Heping Cao; Dorselyn C. Chapital; O. D. Howard Jr.; Leesa J. Deterding; Catherine B. Mason; Jay M. Shockey; K. Thomas Klasson (pp. 711-727).

Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.

Keywords: Diacylglycerol acyltransferase; Protein expression; Protein purification; E. coli (Escherichia coli); Tung tree (Vernicia fordii)

A genetically engineered protein domain binding to bacterial murein, archaeal pseudomurein, and fungal chitin cell wall material by Ganesh Ram R. Visweswaran; Bauke W. Dijkstra; Jan Kok (pp. 729-737).

The major murein and pseudomurein cell wall-binding domains, i.e., the Lysin Motif (LysM) (Pfam PF01476) and pseudomurein cell wall-binding (PMB) (Pfam PF09373) motif, respectively, were genetically fused. The fusion protein is capable of binding to both murein- and pseudomurein-containing cell walls. In addition, it also binds to chitin, the major polymer of fungal cell walls. Binding is influenced by pH and occurs at a pH close to the pI of the binding protein. Functional studies on truncated versions of the fusion protein revealed that murein and chitin binding is provided by the LysM domain, while binding to pseudomurein is achieved through the PMB domain.

Keywords: Murein; Pseudomurein; Chitin; Domain

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis by Hsu-Hua Yeh; Yi-Ming Chiang; Ruth Entwistle; Manmeet Ahuja; Kuan-Han Lee; Kenneth S. Bruno; Tung-Kung Wu; Berl R. Oakley; Clay C. C. Wang (pp. 739-748).

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

Keywords: Aspergillus nidulans ; Nonribosomal peptide synthetase-like; Microperfuranone; Biosynthesis

The redox-sensing protein Rex, a transcriptional regulator of solventogenesis in Clostridium acetobutylicum by Mandy Wietzke; Hubert Bahl (pp. 749-761).

Solventogenic clostridia are characterised by their biphasic fermentative metabolism, and the main final product n-butanol is of particular industrial interest because it can be used as a superior biofuel. During exponential growth, Clostridium acetobutylicum synthesises acetic and butyric acids which are accompanied by the formation of molecular hydrogen and carbon dioxide. During the stationary phase, the solvents acetone, butanol and ethanol are produced. However, the molecular mechanisms of this metabolic switch are largely unknown so far. In this study, in silico, in vitro and in vivo analyses were performed to elucidate the function of the CAC2713-encoded redox-sensing transcriptional repressor Rex and its role in the solventogenic shift of C. acetobutylicum ATCC 824. Electrophoretic mobility shift assays showed that Rex controls the expression of butanol biosynthetic genes as a response to the cellular NADH/NAD⁺ ratio. Interestingly, the Rex-negative mutant C. acetobutylicum rex::int(95) produced high amounts of ethanol and butanol, while hydrogen and acetone production were significantly reduced. Both ethanol and butanol (but not acetone) formation started clearly earlier than in the wild type. In addition, the rex mutant showed a de-repression of the bifunctional aldehyde/alcohol dehydrogenase 2 encoded by the adhE2 gene (CAP0035) as demonstrated by increased adhE2 expression as well as high NADH-dependent alcohol dehydrogenase activities. The results presented here clearly indicated that Rex is involved in the redox-dependent solventogenic shift of C. acetobutylicum.

Keywords: Biofuel; Butanol; ABE fermentation; Redox regulation; ClosTron

The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris by Qi Shen; Ming Wu; Hai-Bin Wang; Hua Naranmandura; Shu-Qing Chen (pp. 763-772).

Interleukin-1 receptor antagonist (IL1ra) is known to treat a number of diseases such as rheumatoid arthritis and type 2 diabetes. However, the biological half-life of IL1ra is very short due to its rapid renal clearance. Our present study aimed to increase the biological half-life of IL1ra through fusion with human serum albumin (HSA), and then augmented expression of the IL1ra and HSA fusion protein (IH) in Pichia pastoris strain by increasing IH gene copy number or was co-expressed with chaperone. By comparing clones containing varying copy numbers of IH fusion gene, it was observed that higher levels of secretory IH fusion protein was produced in strain with higher IH gene copy number. In addition, IH protein yield was further improved after being co-expressed with protein disulfide isomerase (PDI). Conversely, it was significantly decreased (i.e., secretory IH in the culture medium) by co-expression of immunoglobulin binding protein. We have also discussed whether the multi-copy strain and co-expressed of PDI could enhance the levels of other secretory albumin fusion protein (e.g., HSA and human growth hormone fusion protein). Interestingly, the level of this fusion protein was apparently also increased by these approaches. In conclusion, our results have demonstrated that increasing copy number and co-expression of PDI may raise yield of albumin fusion protein in P. pastoris, which might probably contribute to the industry for the development of proteinous drugs.

Keywords: P. pastoris ; Albumin fusion protein; Multi-copy; PDI; BiP

γ-Heptalactone is an endogenously produced quorum-sensing molecule regulating growth and secondary metabolite production by Aspergillus nidulans by Headley E. Williams; Jonathan C. P. Steele; Mark O. Clements; Tajalli Keshavarz (pp. 773-781).

Microbes monitor their population density through a mechanism termed quorum sensing. It is believed that quorum-sensing molecules diffuse from the microbial cells and circulate in the surrounding environment as a function of cell density. When these molecules reach a threshold concentration, the gene expression of the entire population is altered in a coordinated manner. This work provides evidence that Aspergillus nidulans produces at least one small diffusible molecule during its growth cycle which accumulates at high cell density and alters the organism's behaviour. When added to low-density cell cultures, ethyl acetate extracts from stationary phase culture supernatants of A. nidulans resulted in the abolition of the lag phase, induced an earlier deceleration phase with 16.3 % decrease in the final cell dry weight and resulted in a 37.8 % increase in the expression of ipnA::lacZ reporter gene construct, which was used as a marker for penicillin production compared to non-treated controls. The bioactive molecule present in the stationary phase extract was purified to homogeneity and was identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy to be γ-heptalactone. This study provides the first evidence that A. nidulans produces γ-heptalactone at a high cell density and it can alter the organism's behaviour at a low cell density. γ-Heptalactone hence acts as a quorum-sensing molecule in the producing strain.

Keywords: Quorum-sensing molecule; Aspergillus nidulans ; γ-Heptalactone; Penicillin; Growth phase regulation

α-Ketoglutaric acid production by Yarrowia lipolytica and its regulation by Svetlana V. Kamzolova; Maria N. Chiglintseva; Julia N. Lunina; Igor G. Morgunov (pp. 783-791).

The yeast Yarrowia lipolytica VKM Y-2412 was selected as a prospective producer of α-ketoglutaric acid (KGA) from ethanol. The following peculiarities were found: (1) the intensive KGA production occurred only under the limitation of cell growth by thiamine and the excess of ethanol and nitrogen, (2) the production of KGA from ethanol required increased amount of zinc and iron ions, and (3) KGA production increased significantly with a high aeration at pH medium equal to 3.5. Under optimal conditions, the Y. lipolytica VKM Y-2412 produced up to 172 g l⁻¹ of KGA with the mass yield coefficient of 0.70 g g⁻¹.

Keywords: Microbial production; α-Ketoglutaric acid (KGA); Yarrowia lipolytica ; Ethanol; Optimization

A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil by Yong Wang; Kazunari Nagaoka; Masahito Hayatsu; Yoriko Sakai; Kanako Tago; Susumu Asakawa; Takeshi Fujii (pp. 793-802).

The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction–denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction–denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined.

Keywords: Soil; RNA extraction; Autoclaved casein; Andosol; amoA

Growth characteristics of three Fusarium species evaluated by near-infrared hyperspectral imaging and multivariate image analysis by Paul J. Williams; Paul Geladi; Trevor J. Britz; Marena Manley (pp. 803-813).

Colony growth of three Fusarium spp. on potato dextrose agar was followed by collecting near-infrared (NIR) hyperspectral images of the colonies at regular intervals after inoculation up to 55 h. After principal component analysis (PCA), two clusters were apparent in the score plot along principal component 1. Using the brushing technique, these clusters were divided into four groups of pixels with similar score values. These could be visualised as growth zones within the colonies in the corresponding score image. Three spectral bands, i.e. 1,166, 1,380 and 1,918 nm, were prominent in the multiplicative scatter corrected and Savitzky–Golay second derivative spectra. These indicated chemical changes, associated with carbohydrates (1,166 and 1,380 nm) and protein (1,918 nm), that occurred as the mycelium grew and matured. The protein band was more prominent in the mature fungal material while the carbohydrate band was less pronounced. The younger material and the agar were characterised by the carbohydrate spectral band. Integrating whole mycelium colonies as the sum of pixels over time made it possible to construct curves that resembled growth curves; this included the lag phase, active growth phase, deceleration phase and phase of constant growth. Growth profiles constructed from individual growth zones indicated more detailed growth characteristics. The use of NIR hyperspectral imaging and multivariate image analysis (MIA) allowed one to visualise radial growth rings in the PCA score images. This would not have been possible with bulk spectroscopy. Interpreting spectral data enabled better understanding of microbial growth characteristics on agar medium. NIR hyperspectral imaging combined with MIA is a powerful tool for the evaluation of growth characteristics of fungi.

Keywords: Near-infrared hyperspectral imaging; Microbial growth characteristics; Fusarium ; PCA; Time-dependent imaging

Backwash intensity and frequency impact the microbial community structure and function in a fixed-bed biofilm reactor by Xu Li; Wangki Yuen; Eberhard Morgenroth; Lutgarde Raskin (pp. 815-827).

Linkages among bioreactor operation and performance and microbial community structure were investigated for a fixed-bed biofilm system designed to remove perchlorate from drinking water. Perchlorate removal was monitored to evaluate reactor performance during and after the frequency and intensity of the backwash procedure were changed, while the microbial community structure was studied using clone libraries and quantitative PCR targeting the 16S rRNA gene. When backwash frequency was increased from once per month to once per day, perchlorate removal initially deteriorated and then recovered, and the relative abundance of perchlorate-reducing bacteria (PRB) initially increased and then decreased. This apparent discrepancy suggested that bacterial populations other than PRB played an indirect role in perchlorate removal, likely by consuming dissolved oxygen, a competing electron acceptor. When backwash intensity was increased, the reactor gradually lost its ability to remove perchlorate, and concurrently the relative abundance of PRB decreased. The results indicated that changes in reactor operation had a profound impact on reactor performance through altering the microbial community structure. Backwashing is an important yet poorly characterized procedure when operating fixed-bed biofilm reactors. Compared to backwash intensity, changes in backwash frequency exerted less disturbance on the microbial community in the current study. If this finding can be confirmed in future work, backwash frequency may serve as the primary parameter when optimizing backwash procedures.

Keywords: Backwash frequency; Backwash intensity; Microbial community; Biofilm reactor; Perchlorate

Biosorption behavior and mechanism of heavy metals by the fruiting body of jelly fungus (Auricularia polytricha) from aqueous solutions by Haiwei Huang; Lixiang Cao; Yuxuan Wan; Renduo Zhang; Wenfeng Wang (pp. 829-840).

The aim of this study was to investigate the biosorption characteristics of Cd²⁺, Cu²⁺, and Pb²⁺ by the fruiting body of jelly fungus Auricularia polytricha. Batch experiments were conducted to characterize the kinetics, equilibrium, and mechanisms of the biosorption process. Optimum values of pH 5, biomass dosage 4 g L⁻¹, and contact time 60 min provided maximum biosorption capacities of A. polytricha for Cd²⁺, Cu²⁺, and Pb²⁺ of 63.3, 73.7, and 221 mg g⁻¹, respectively. The maximum desorption was achieved using 0.05 mol L⁻¹ HNO₃ as an elute. The fruiting body was reusable at least for six cycles of operations. The pseudo-second-order model was the best to describe the biosorption processes among the three kinetic models tested. Freundlich and Dubinin–Radushkevich models fitted the equilibrium data well, indicating a heterogeneous biosorbent surface and the favorable chemisorption nature of the biosorption process. A Fourier transform infrared spectroscopy analysis indicated that carboxyl, amine/hydroxyl, amino, phosphoryl, and C–N–C were the main functional groups to affect the biosorption process. Synergistic ion exchange and surface complexation were the dominant mechanisms in the biosorption process. The present work revealed the potential of jelly fungus (fruiting body of A. polytricha) to remove toxic heavy metals from contaminated water.

Keywords: Biosorption; Jelly fungus; Auricularia ploytricha ; Heavy metals

Prolongation of electrode lifetime in biofuel cells by periodic enzyme renewal by S. Rubenwolf; S. Sané; L. Hussein; J. Kestel; F. von Stetten; G. Urban; M. Krueger; R. Zengerle; S. Kerzenmacher (pp. 841-849).

Enzymatically catalyzed biofuel cells show unique specificity and promise high power densities, but suffer from a limited lifetime due to enzyme deactivation. In the present work, we demonstrate a novel concept to extend the lifetime of a laccase-catalyzed oxygen reduction cathode in which we decouple the electrode lifetime from the limited enzyme lifetime by a regular resupply of fresh enzymes. Thereto, the adsorption behavior of laccase from Trametes versicolor to buckypaper electrode material, as well as its time-dependent deactivation characteristics, has been investigated. Laccase shows a Langmuir-type adsorption to the carbon nanotube-based buckypaper electrodes, with a mean residence time of 2 days per molecule. In a citrate buffer of pH 5, laccase does not show any deactivation at room temperature for 2 days and exhibits a half-life of 9 days. In a long-term experiment, the laccase electrodes were operated at a constant galvanostatic load. The laccase-containing catholyte was periodically exchanged against a freshly prepared one every second day to provide sufficient active enzymes in the catholyte for the replacement of desorbed inactive enzymes. Compared to a corresponding control experiment without catholyte exchange, this procedure resulted in a 2.5 times longer cathode lifetime of 19 ± 9 days in which the electrode showed a potential above 0.744 V vs. normal hydrogen electrode at 110 μA cm⁻². This clearly indicates the successful exchange of molecules by desorption and re-adsorption and is a first step toward the realization of a self-regenerating enzymatic biofuel cell in which enzyme-producing microorganisms are integrated into the electrode to continuously resupply fresh enzymes.

Keywords: Enzymatic biofuel cell; Laccase; Lifetime; Long-term stability; Adsorption; Buckypaper

Erratum to: The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris by Qi Shen; Min Wu; Hai-Bin Wang; Hua Naranmandura; Shu-Qing Chen (pp. 851-851).

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Applied Microbiology and Biotechnology (v.96, #3)

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Applied Microbiology and Biotechnology (v.96, #3)