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A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 16777

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Dr. Seraphim Papanikolaou

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Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
Interests: fermentation biotechnology; kinetics and biochemistry of accumulation and degradation of storage lipids by molds and yeasts; biotechnological valorization of agro-industrial wastes and residues; production and study of microbial metabolic products (microbial lipids, citric acid, 1,3-propanediol, enzymes, 2,3-butanediol)
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Dr. Panagiota Diamantopoulou

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Laboratory of Edible Fungi, Institute of Technology of Agricultural Products, Hellenic Agricultural Organization-Dimitra, 1, Sof. Venizelou, 14123 Lykovrysi, Greece
Interests: mushroom biotechnology; solid-state fermentations by fungi; liquid cultures of higher and lower fungi; fungal metabolites; waste and biomass valorization
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The current Special Issue will present research dealing with the production of several types of metabolites, performed by both eukaryotic (i.e., fungi, yeasts, algae) and prokaryotic (i.e., bacteria, cyanobacteria, archaea) microbial strains. Low-value (i.e., biogas, compost) and high-value (i.e., speciality polysaccharides, polyols, speciality fatty acids) metabolic products, as well as microbial compounds of interest to the food, chemical, pharmaceutical, and biofuel industries will be considered in this Special Issue. Papers dealing with aspects of applied biochemistry, fermentation technology, biochemical engineering, and molecular biology related to the synthesis and production of metabolites performed by all types of the previously mentioned microorganisms will also be of interest. An additional important focus of this Special Issue will be the microbial conversions of residues and low-cost materials.

Metabolic compounds that are of particular interest in this Special Issue include, but are not limited to, proteins, organic acids (i.e., citric acid, oxalic acid, etc.), polyols (i.e., mannitol, erythritol, arabitol, etc.), ethanol, enzymes, microbial lipids, microbial polysaccharides, biohydrogen, and biogas, etc.

All submitted papers will be subjected to the standard independent peer-review process. Mini-reviews are more than welcome in this Special Issue.

Dr. Seraphim Papanikolaou
Dr. Panagiota Diamantopoulou
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Microorganisms is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

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Research

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16 pages, 2533 KiB

Open AccessArticle

Effects of Vachellia mearnsii Tannin Extract as an Additive on Fermentation Quality, Aerobic Stability, and Microbial Modulation of Maize Silage

by Thamsanqa Doctor Empire Mpanza and Sinalo Mani

Microorganisms 2023, 11(11), 2767; https://doi.org/10.3390/microorganisms11112767 - 14 Nov 2023

Cited by 2 | Viewed by 792

Abstract

Maize silage is produced to alleviate the effects of forage shortages on ruminant animals, particularly during the dry season. Microorganisms play a significant role in silage fermentation and thus, to a large extent, determine the silage quality. The modulation of silage microorganisms may help to inhibit undesirable bacteria and improve the silage quality. Therefore, condensed tannin extract from Vachellia mearnsii bark was used as an additive in maize silage during ensiling. Hence, this study evaluated the effects of a tannin extract (condensed tannin) additive on the fermentative quality, aerobic stability, and bacterial composition of maize silage. A mini-silo experiment on maize with five treatments was conducted for 75 days. The silage treatments were as follows: (T1) maize forage with no inoculation (negative control); (T2) maize forage inoculated with LAB and 1% tannin extract; (T3) maize forage inoculated with LAB only (positive control); (T4) and maize forage inoculated with LAB and 2% tannin extract; (T5) maize forage inoculated with LAB and 3% tannin extract. The results showed that the additives modulated the silage microorganism composition. However, this was without affecting the silage’s fermentative quality and aerobic stability. All the silages recorded a pH below 4.2, which indicated well-fermented silage. The tannin extract suppressed the growth of undesirable bacteria, such as Dysgonomonas, Gluconacetobacter and Clostridium genera, while promoting desirable bacteria, such as Lactobacillus and Weissella genera, which were attributed to the silage quality. It is thus concluded that tannins can be strategically used as silage additives to modulate the microbial composition of silage and improve the silage quality by promoting the dominance of the desirable bacteria in the silage. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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24 pages, 1996 KiB

Open AccessArticle

Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 2: Citric Acid Production and Circular-Oriented Valorization of Glucose-Enriched Olive Mill Wastewaters Using Novel Yarrowia lipolytica Strains

by Dimitris Sarris, Erminta Tsouko, Angelos Photiades, Sidoine Sadjeu Tchakouteu, Panagiota Diamantopoulou and Seraphim Papanikolaou

Microorganisms 2023, 11(9), 2243; https://doi.org/10.3390/microorganisms11092243 - 06 Sep 2023

Cited by 1 | Viewed by 1048

Abstract

The global market for citric acid (CA) is one of the biggest and fastest expanding markets in the food industry. The CA production employing microbial bioprocessing with efficient GRAS strains and renewable waste streams is in line with the European Union binding targets for resource efficiency, sustainable consumption-production, and low-carbon technologies. In this work, the potential of three novel wild-type Yarrowia lipolytica strains (namely LMBF Y-46, LMBF Y-47 and ACA-YC 5033) regarding the production of CA and other valuable metabolites was tested on glucose-based media, and the most promising amongst the screened strains (viz. the strain ACA-YC 5033) was cultured on glucose-based media, in which part of the fermentation water had been replaced by olive-mill wastewaters (OMWs) in a novel approach of simultaneous OMW valorization and bioremediation. In the first part of this study, the mentioned strains were cultured under nitrogen-limited conditions with commercial (low-cost) glucose employed as a sole carbon source in shake-flask cultures at an initial concentration (S₀) ≈ of 50 g/L. Variable quantities of secreted citric acid (CA) and intra-cellular compounds (viz. polysaccharides and lipids) were produced. All strains did not accumulate significantly high lipid quantities (i.e., maximum lipid in dry cell weight [DCW] values ≈30% w/w were noted) but produced variable CA quantities. The most promising strain, namely ACA-YC 5033, produced CA up to c. 24 g/L, with a yield of CA produced on glucose consumed (Y_CA/S) ≈ 0.45 g/g. This strain in stirred tank bioreactor experiments, at remarkably higher S₀ concentrations (≈110 g/L) and the same initial nitrogen quantity added into the medium, produced notably higher CA quantities, up to 57 g/L (Y_CA/S ≈ 0.52 g/g). The potential of the same strain (ACA-YC 5033) to bioremediate OMWs and to produce value-added compounds, i.e., yeast cells, CA, and intra-cellular metabolites, was also assessed; under nitrogen-limited conditions in which OMWs had partially replaced tap water and significant glucose concentrations had been added (S₀ ≈ 100 g/L, simultaneous molar ratio C/N ≈ 285 g/g, initial phenolic compounds [Phen₀] adjusted to ≈1.0 g/L; these media were similar to the OMWs generated from the traditional press extraction systems) the notable CA quantity of 60.2 g/L with simultaneous Y_CA/S = 0.66 g/g, was obtained in shake flasks, together with satisfactory phenolic compounds removal (up to 19.5% w/w) and waste decolorization (up to 47.0%). Carbon-limited conditions with Phen₀ ≈ 1.0 g/L favored the production of yeast DCW (up to 25.3 g/L), with equally simultaneous interesting phenolic compounds and color removal. The fatty acid profile showed that cellular lipids were highly unsaturated with oleic, linoleic and palmitoleic acids, accounting for more than 80% w/w. This study proposed an interesting approach that could efficiently address the biotreatment of toxic effluents and further convert them into circular-oriented bioproducts. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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13 pages, 2167 KiB

Open AccessArticle

The Quantitative Measurement of Peptidoglycan Components Obtained from Acidic Hydrolysis in Gram-Positive and Gram-Negative Bacteria via Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry

by Dmitri Pismennõi, Anna Kattel, Isma Belouah, Ranno Nahku, Raivo Vilu and Eeva-Gerda Kobrin

Microorganisms 2023, 11(9), 2134; https://doi.org/10.3390/microorganisms11092134 - 23 Aug 2023

Viewed by 1352

Abstract

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes’ prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram− bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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18 pages, 1141 KiB

Open AccessArticle

Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 1: High Production of Lipid by Lipomyces starkeyi and Citric Acid by Yarrowia lipolytica

by Panagiota Diamantopoulou, Dimitris Sarris, Sidoine Sadjeu Tchakouteu, Evangelos Xenopoulos and Seraphim Papanikolaou

Microorganisms 2023, 11(7), 1863; https://doi.org/10.3390/microorganisms11071863 - 24 Jul 2023

Cited by 6 | Viewed by 1188

Abstract

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very “hot” scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired (“waste”) glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to “boost” the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc₀) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc₀ concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc₀ = 85 g/L, produced DCW_max = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc₀ ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; Y_CA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (Y_CA/Glc = 0.50 g/g) was recorded. Concluding, “waste” glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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18 pages, 2015 KiB

Open AccessArticle

Screening of New Industrially Important Bacterial Strains for 1,3-Propanediol, 2,3-Butanediol and Ethanol Production through Biodiesel-Derived Glycerol Fermentations

by Dimitris Karayannis, Gabriel Vasilakis, Ioannis Charisteidis, Alexandros Litinas, Eugenia Manolopoulou, Effie Tsakalidou and Seraphim Papanikolaou

Microorganisms 2023, 11(6), 1424; https://doi.org/10.3390/microorganisms11061424 - 28 May 2023

Cited by 2 | Viewed by 1586

Abstract

A study on the ability of new microbial strains to assimilate biodiesel-derived glycerol at low purity (75% w/w) and produce extra-cellular platform chemical compounds of major interest was carried out. After screening several bacterial strains under different fermentation conditions (e.g., pH, O₂ availability, glycerol purity), three of the screened strains stood out for their high potential to produce valued-added products such as 2,3-butanediol (BDO), 1,3-propanediol (PDO) and ethanol (EtOH). The results indicate that under aerobic conditions, Klebsiella oxytoca ACA-DC 1581 produced BDO in high yield (Y_BDO/Gly = 0.46 g/g, corresponding to 94% of the maximum theoretical yield; Y_mt) and titer, while under anaerobic conditions, Citrobacter freundii NRRL-B 2645 and Enterobacter ludwigii FMCC-204 produced PDO (Y_PDO/Gly = 0.56 g/g, 93% of Y_mt) and EtOH (Y_EtOH/Gly = 0.44 g/g, 88% of Y_mt), respectively. In the case of C. freundii, the regulation of pH proved to be mandatory, due to lactic acid production and a subsequent drop of pH that resulted in fermentation ceasing. In the fed-batch culture of K. oxytoca, the BDO maximum titer reached almost 70 g/L, the Y_BDO/Gly and the mean productivity value (Pr_BDO) were 0.47 g/g and 0.4 g/L/h, respectively, while no optimization was imposed. The final BDO production obtained by this wild strain (K. oxytoca) is among the highest in the international literature, although the bioprocess requires optimization in terms of productivity and total cost. In addition, for the first time in the literature, a strain from the species Hafnia alvei (viz., Hafnia alvei ACA-DC 1196) was reported as a potential BDO producer. The strains as well as the methodology proposed in this study can contribute to the development of a biorefinery that complements the manufacture of biofuels with high-value biobased chemicals. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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13 pages, 1632 KiB

Open AccessArticle

The Role of the PAA1 Gene on Melatonin Biosynthesis in Saccharomyces cerevisiae: A Search of New Arylalkylamine N-Acetyltransferases

by Ricardo Bisquert, Andrés Planells-Cárcel, Javier Alonso-del-Real, Sara Muñiz-Calvo and José Manuel Guillamón

Microorganisms 2023, 11(5), 1115; https://doi.org/10.3390/microorganisms11051115 - 25 Apr 2023

Viewed by 1365

Abstract

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate’s aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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17 pages, 1737 KiB

Open AccessArticle

Spent Mushroom Substrate Hydrolysis and Utilization as Potential Alternative Feedstock for Anaerobic Co-Digestion

by Gabriel Vasilakis, Evangelos-Markos Rigos, Nikos Giannakis, Panagiota Diamantopoulou and Seraphim Papanikolaou

Microorganisms 2023, 11(2), 532; https://doi.org/10.3390/microorganisms11020532 - 20 Feb 2023

Cited by 2 | Viewed by 2420

Abstract

Valorization of lignocellulosic biomass, such as Spent Mushroom Substrate (SMS), as an alternative substrate for biogas production could meet the increasing demand for energy. In view of this, the present study aimed at the biotechnological valorization of SMS for biogas production. In the first part of the study, two SMS chemical pretreatment processes were investigated and subsequently combined with thermal treatment of the mentioned waste streams. The acidic chemical hydrolysate derived from the hydrothermal treatment, which yielded in the highest concentration of free sugars (≈36 g/100 g dry SMS, hydrolysis yield ≈75% w/w of holocellulose), was used as a potential feedstock for biomethane production in a laboratory bench-scale improvised digester, and 52 L biogas/kg of volatile solids (VS) containing 65% methane were produced in a 15-day trial of anaerobic digestion. As regards the alkaline hydrolysate, it was like a pulp due to the lignocellulosic matrix disruption, without releasing additional sugars, and the biogas production was delayed for several days. The biogas yield value was 37 L/kg VS, and the methane content was 62%. Based on these results, it can be concluded that SMS can be valorized as an alternative medium employed for anaerobic digestion when pretreated with both chemical and hydrothermal hydrolysis. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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Review

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21 pages, 1805 KiB

Open AccessReview

Adaptive Laboratory Evolution of Microorganisms: Methodology and Application for Bioproduction

by Takashi Hirasawa and Tomoya Maeda

Microorganisms 2023, 11(1), 92; https://doi.org/10.3390/microorganisms11010092 - 29 Dec 2022

Cited by 12 | Viewed by 5148

Abstract

Adaptive laboratory evolution (ALE) is a useful experimental methodology for fundamental scientific research and industrial applications to create microbial cell factories. By using ALE, cells are adapted to the environment that researchers set based on their objectives through the serial transfer of cell populations in batch cultivations or continuous cultures and the fitness of the cells (i.e., cell growth) under such an environment increases. Then, omics analyses of the evolved mutants, including genome sequencing, transcriptome, proteome and metabolome analyses, are performed. It is expected that researchers can understand the evolutionary adaptation processes, and for industrial applications, researchers can create useful microorganisms that exhibit increased carbon source availability, stress tolerance, and production of target compounds based on omics analysis data. In this review article, the methodologies for ALE in microorganisms are introduced. Moreover, the application of ALE for the creation of useful microorganisms as cell factories has also been introduced. Full article

(This article belongs to the Special Issue Advances in Microbial Metabolites)

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