Appl. Microbiol., Volume 2, Issue 1 (March 2022) – 21 articles

Biological control is an environmentally friendly approach that holds promise to complement or replace chemicals to effectively protect crop plants against pests and pathogens. Environmental samples with highly diverse and competitive microbiomes that harbor antagonistic microbes with diverse modes-of-action can provide a rich source of microbial biopesticides. In the current study, bacteria isolated from rhizosphere soil and food spoilage samples were subsequently screened against various plant fungal and oomycete pathogens in growth inhibition assays. These included the new potential biocontrol bacteria Corynebacterium flavescens, Sporosarcina aquimarina and Sporosarcina saromensis with anti-fungal and antioomycete activities. Potential candidates selected by preliminary screening in plant assays were then applied to tomato, cabbage and chickpea plants to control bacterial (Pseudomonas syringae pv. tomato), fungal (Alternaria brassicicola) and oomycete (Phytophtora medicaginis) phytopathogens. Ten potential microbial biopesticides were demonstrated to be effective against these diseases, and led to significant (p < 0.05) reductions in symptoms and/or pathogen DNA compared to mock-treated diseased plants. We conclude that new and effective microbial biopesticides to control crop pathogens can be rapidly isolated from biodiverse microbiomes, where bacteria may employ these features to effectively compete against each other. Full article

Bioregenerative life support systems (BLSS) are currently in development to tackle low recovery efficiencies, high energy demands, as well as food, water, and oxygen production challenges through the regeneration of nutrients from waste streams. The MELiSSA pilot plant has been developed as a testbed for regenerative life support system bioreactor operation and characterization. As nitrogen is a vital resource in such systems, we studied the functional composition of a new packed-bed nitrifying bioreactor inoculated with a co-culture of Nitrosomonas europaea (ATCC 25978) and Nitrobacter winogradskyi (ATCC 25391). After 840 days of autotrophic continuous cultivation, the packed-bed was sampled at five vertical positions, each with three horizontal positions, and the biomass at each position was characterized via qPCR, 16S amplicon sequencing, and liquid chromatography tandem mass spectrometry. The total number of cells within the different sections fluctuated around 8.95 ± 5.10 × 10⁷ cells/mL of beads. Based on 16S amplicons and protein content, N. europaea and N. winogradskyi constituted overall 44.07 ± 11.75% and 57.53 ± 12.04% of the nitrifying bioreactor, respectively, indicating the presence of a heterotrophic population that, even after such a long operation time, did not affect the nitrification function of the bioreactor. In addition, DNA-based abundance estimates showed that N. europaea was slightly more abundant than N. winogradskyi, whereas protein-based abundance estimates indicated a much higher abundance of N. europaea. This highlights that single-method approaches need to be carefully interpreted in terms of overall cell abundance and metabolic activity. Full article

This novel study investigated the loading of mupirocin nanoparticles into a hydrogel which was expected to enhance the antibacterial activity of mupirocin. The inhibition of isoleucyl-tRNA synthetase and global gene expression in methicillin-resistant Staphylococcus aureus (MRSA) by the mupirocin nanoparticle-loaded hydrogel (MLH) and by pure mupirocin was compared. MLH and mupirocin rapidly inhibited the growth of bacterial populations after 1 h of treatment. At 12 h, mupirocin and MLH inhibited isoleucyl-tRNA synthetase in MRSA. Transcriptome profiles of MRSA showed that gene expression alterations after treatment with mupirocin were similar to those after treatment with MLH at MICs. These alterations included changes to DNA transcription, translation, and replication pathways, and the fold changes in these genes decreased more rapidly with MLH than with mupirocin only after 1 h of treatment. MLH released the mupirocin from the nanoparticles and hydrogel systems and then the drug permeated the cell wall and bound to bacterial isoleucyl-tRNA synthetase. The research also showed that MLH could be further developed for use in clinics for infected wounds. Full article

Biosurfactants are amphiphilic molecules with surface tension reducing activities. Among biosurfactant producers, fungi have been identified as promising organisms. While many studies have investigated biosurfactant production in fungal species from the Ascomycota and Basidiomycota phyla, less is known concerning species from the Mucoromycota phylum. In this context, the aim of this study was to screen and optimize biosurfactant production in 24 fungal strains, including seven Mucor, three Lichtheimia, and one Absidia species. After cultivation in a medium stimulating surfactant production, the surface activity of cell-free supernatants was measured using both oil spreading and parafilm M tests. Among them, five Mucor strain cell-free supernatants belonging to M. circinelloides, M. lanceolatus, M. mucedo, M. racemosus, and M. plumbeus, showed oil repulsion. Then, the impact of the medium composition on surfactant production was evaluated for eight strains. Three of them, i.e., Mucor circinelloides UBOCC-A-109190, Mucor plumbeus UBOCC-A-111133, and Mucor mucedo UBOCC-A-101353 showed an interesting surfactant production potential, reducing the medium surface tension to 36, 31, and 32 mN/m, respectively. A preliminary characterization of the surfactant molecules produced by these strains was performed and showed that these compounds belonged to the glycolipid family. Full article

Microbial lipase is one of the major sources of the enzyme that has been broadly exploited in the food, detergent, and pharmaceutical industries due to its high catalytic activity, high yield, and environmental friendliness and cost-effectiveness. Therefore, the aim of this study was to optimize the medium for the submerged fermentation for lipase production by a novel strain, Bacillus salmalaya strain 139SI. The media subjected to lipase production was Luria Bertani (LB) with different carbon sources and surfactants supplemented to determine which would give the highest lipase activity of Bacillus salmalaya. The Lipase activity of the supernatant containing lipase enzyme was ddetermined using the titrimetric method with hydrolysis reaction. Results showed that the olive oil that was used as a carbon source, induced the highest lipase activity (11.0 U/mL) compared to sunflower oil (9.6 U/mL) and cooking oil waste (7.8 U/mL). For surfactants, LB medium supplemented with tween 80 enhanced higher lipase activity (6.8 U/mL) compared to tween 20 (6.0 U/mL) and sodium dodecyl sulphate (SDS) (2.0 U/mL). Thus, it can be concluded that submerged fermentation allows optimization of the culture medium whereby, among carbon sources, olive oil induced the highest lipase production, whereas Tween 80 was the best lipase inducer compared to other surfactants. Full article

Microbial infections, and especially microbial resistance, are critical and actual problems that require targeted and efficient therapeutic intervention. Natural-based solutions are a viable alternative, at least for complementary therapy, due to few or no side effects and high safety and efficacy levels. The aim of this study was to demonstrate the potential use of a patented formula based on Achillea millefolium, Origanum vulgare, and Lychnis coronaria species as an antibacterial ingredient, mainly for skin and mucosal infections, in order to support its pharmaco-cosmetic application. The chemical composition of the formula was analyzed by HPLC and spectrophotometric methods. Furthermore, antioxidant and antimicrobial activity were evaluated. To determine the formula’s safety for topical application, it was used on a reconstructed human epidermal model. The formula showed inhibitory activity on both Gram-positive and Gram-negative bacteria, respectively, moderate inhibition on B. cereus, Kocuria kristinae, P. aeurginosa, S. enterica Typhimurium, methicillin-resistant and methicillin-sensible S. aureus, as well as high inhibition on S. epidermidis, Serratia marescens, and Streptococcus pyogenes. The developed product was biochemically characterized for its content in polyphenols, triterpenes, and polyphenol carboxylic acids. The formula was proven to have a nonirritant effect on the human epidermis and important antioxidant activity. Full article

As a renewable energy carrier, dark fermentative biohydrogen (H₂) represents a promising future alternative to fossil fuels. Recently, the limited H₂ yield of 4 moles of H₂ per mole glucose, the so-called “Thauer limit”, was surpassed by a defined artificial consortium. In this article, we demonstrate the upscaling of this drawing board design, from serum bottles to laboratory scale bioreactors. Our results illustrate that this designed microbial co-culture can be successfully implemented in batch mode, with maximum H₂ yields of 6.18 and 4.45 mol mol⁻¹ substrate. Furthermore, we report volumetric H₂ productivities of 105.6 and 80.8 mmol H₂ L⁻¹ h⁻¹_. These rates are higher than for any other dark fermentative H₂ production system using a synthetic microbial co-culture applied in batch mode on a defined medium. Our study is an important step forward for the application of artificial microbial consortia in future biotechnology and energy production systems. Full article

Based on the collected data on the diversity of microalgae and environmental indicators in dry and wet seasons during 2011–2018, from 45 samples, 59 species of microalgae were identified in the ornithological object—Lake Agmon in the Hula Valley. In the samples of periphyton and microphytobenthos, diatoms predominated. Bioindication analysis and statistical mapping revealed the most pronounced zones of influence on the lake ecosystem, as well as indicators of the environment and diversity that clearly demonstrate them. The correlation between the distribution of TDS of water over the lake surface and the distribution of green, diatom microalgae and cyanobacteria detected two areas of impact from the old channel of the Jordan River in the northwestern part and from the drainage channel in the northeastern parts of the lake. The area on the east coast, in contact with the resting fields of migratory birds, has provided nutrients that stimulate the development of green algae and cyanobacteria. This showed implicit links in the lake ecosystem using bioindicators make it possible to recommend them for monitoring in combination with statistical mapping, which visualizes the distribution of data and is easily accessible for the decision-making system for the management of a protected ornithological lake. Full article

This study proposes a method for the chamber disinfection of paper with biocide in the form of fine, volatile droplets using antifungal Rocima GT. This method provides a fungicidal effect, and within a short exposure time, a fungistatic one. At a concentration of 5% Rocima GT solution, the minimum treatment time to ensure the complete disinfection of paper was 15 min. The proposed method of disinfection by a chemical mist was less harmful to paper than disinfection using a swab saturated in a biocide solution. It was noted that when using Rocima GT at insufficient concentrations to inhibit fungal growth completely, Rocima GT can induce, as well as suppress, organic acid produced by Aspergillus niger depending on the concentration. Full article

The use of probiotic bacteria can not only enhance the nutritional utilization of fish feeds to produce more biomass but can also provide a practically “safer” alternative to the fish farming industry to reduce the abuse of antibiotics and drugs. This study investigated the possibility of colonizing Lactobacillus rhamnosus strain GG (LGG) to the intestine of Cirrhinus molitorella (mud carp) fingerling. Colonization of LGG was observed in gut tissue after 14 days of administration with a diet supplemented with 1 × 10⁸ CFU/mL LGG. Moreover, growth performance parameters of the LGG-supplemented diet group, including relative weight gain, feed conversion ratio and feed efficiency, were found about two-fold higher than the control group after 60 days. In addition, fish fed with an LGG-supplemented diet for 60 days showed substantial resistance against the infection of pathogenic bacterial Aeromonas hydrophila, with a relative survival rate of up to 57% compared to the control group. In summary, the results indicated that LGG as dietary supplement for mud carp fingerling can enhance nutrition utilization and better protect fish against the infection of Aeromonas hydrophila. The results provide an insight to the fish farming industry, encouraging a reduction in the use of antibiotics and drugs and the production of “safer” mud carp for the market at a manageable cost. Full article

Synthetic biology applications rely on a well-characterized set of microbial strains, with an established toolbox of molecular biology methods for their genetic manipulation. Since there are no thermophiles with such attributes, most biotechnology and synthetic biology studies use organisms that grow in the mesophilic temperature range. As a result, thermophiles, a heterogenous group of microbes that thrive at high (>50 °C) temperatures, are largely overlooked, with respect to their biotechnological potential, even though they share several favorable traits. Thermophilic bacteria tend to grow at higher rates compared to their mesophilic counterparts, while their growth has lower cooling requirements and is less prone to contamination. Over the last few years, there has been renewed interest in developing tools and methods for thermophile bioengineering. In this perspective, we explain why it is a good idea to invest time and effort into developing a thermophilic synthetic biology direction, which is the state of the art, and why we think that the implementation of a thermophilic synthetic biology platform—a thermochassis—will take synthetic biology to the extremes. Full article

Mass spectrometry (MS) is one of the key technologies used in proteomics. The majority of studies carried out using proteomics have focused on identifying proteins in biological samples such as human plasma to pin down prognostic or diagnostic biomarkers associated with particular conditions or diseases. This study aims to quantify microbial (viral and bacterial) proteins in healthy human plasma. MS data of healthy human plasma were searched against the complete proteomes of all available viruses and bacteria. With this baseline established, the same strategy was applied to characterize the metaproteomic profile of different SARS-CoV-2 disease stages in the plasma of patients. Two SARS-CoV-2 proteins were detected with a high confidence and could serve as the early markers of SARS-CoV-2 infection. The complete bacterial and viral protein content in SARS-CoV-2 samples was compared for the different disease stages. The number of viral proteins was found to increase significantly with the progression of the infection, at the expense of bacterial proteins. This strategy can be extended to aid in the development of early diagnostic tests for other infectious diseases based on the presence of microbial biomarkers in human plasma samples. Full article

The aim of this study was to investigate the pathogenic potential and antibiotic resistance of 59 Escherichia coli isolates from ready-to-eat (RTE) street food (n = 31) and drinking water (n = 28) sold in the city of Maputo, Mozambique. The isolates were characterized by XbaI subtyping analysis via pulsed field gel electrophoresis. Multiplex PCRs were performed targeting five virulence genes (stx, lt, st, astA, and eae) and three groups of antibiotic-resistant genes, namely ß-lactamases (extended-spectrum ß-lactamase and AmpC), tetracycline (tetA, tetB, and tetM) and sulfamethoxazole/trimethoprim (sul1, sul2, and sul3). The stx virulence gene, encoding the Shiga/Vero (VT) toxin produced by the verotoxin-producing E. coli (VTEC), was identified with similar frequency in isolates from food (5/31) and water (6/28). The highest percentages of resistant isolates from food and water were found for ß-lactams imipenem (35.5 and 39.3%, respectively) and ampicillin (39.3 and 46.4%, respectively). Multidrug resistance was observed in 31.3% of the isolates, being higher in E. coli isolates from water (45.5%) compared to RTE street food isolates (19.2%). Virulence genes were detected in 73% of the multidrug-resistant isolates. Concerning antibiotic-resistant genes, ESBL was the most frequent (57.7%) among β-lactamases while tetA was the most frequent (50%) among non-β-lactamases. Full article

The aim of this study was the biochemical and kinetic characterization of the gentisate 1,2-dioxygenase (GDO) from Pseudarthrobacter phenanthrenivorans Sphe3 and the development of a nanobiocatalyst by its immobilization on Ni²⁺-functionalized Fe₃O₄-polydopamine magnetic nanoparticles (Ni²⁺-PDA-MNPs). This is the first GDO to be immobilized. The gene encoding the GDO was cloned with an N-terminal His-tag and overexpressed in E. coli. The nanoparticles showed a high purification efficiency of GDO from crude cell lysates with a maximum activity recovery of 97%. The immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR). The reaction product was identified by ¹H NMR. Both free and immobilized GDO exhibited Michaelis–Menten kinetics with K_m values of 25.9 ± 4.4 and 82.5 ± 14.2 μM and V_max values of 1.2 ± 0.1 and 0.03 ± 0.002 mM·s⁻¹, respectively. The thermal stability of the immobilized GDO was enhanced at 30 °C, 40 °C, and 50 °C, compared to the free GDO. Stored at −20 °C, immobilized GDO retained more than 60% of its initial activity after 30 d, while the free enzyme completely lost its activity after 10 d. Furthermore, the immobilized nanoparticle–enzyme conjugate retained more than 50% enzyme activity up to the fifth cycle. Full article

Benzophenone-3 (BP3) is an organic UV filter whose presence in the aquatic environment has been linked to detrimental developmental impacts in aquatic organisms such as coral and fish. The genus Rhodococcus has been extensively studied and is known for possessing large genomes housing genes for biodegradation of a wide range of compounds, including aromatic carbons. Here, we present the genome sequence of Rhodococcus sp. USK10, which was isolated from Chinese riverbank sediment and is capable of utilising BP3 as the sole carbon source, resulting in full BP3 mineralisation. The genome consisted of 9,870,030 bp in 3 replicons, a G+C content of 67.2%, and 9722 coding DNA sequences (CDSs). Annotation of the genome revealed that 179 of these CDSs are involved in the metabolism of aromatic carbons. The complete genome of Rhodococcus sp. USK10 is the first complete, annotated genome sequence of a Benzophenone-3-degrading bacterium. Through radiolabelling, it is also the first bacterium proven to mineralise Benzophenone-3. Due to the widespread environmental prevalence of Benzophenone-3, coupled with its adverse impact on aquatic organisms, this characterisation provides an integral first step in better understanding the environmentally relevant degradation pathway of the commonly used UV filter. Given USK10′s ability to completely mineralise Benzophenone-3, it could prove to be a suitable candidate for bioremediation application. Full article

Kombucha is a carbonated, slightly acidic beverage traditionally produced by the fermentation of sweetened tea by a symbiotic culture of bacteria and yeast (SCOBY). The microbial community of kombucha is a complex one, whose dynamics are still not fully understood; however, the emergence of culture-independent techniques has allowed a more comprehensive insight into kombucha microbiota. In recent times, advancements have been made towards the optimisation of the fermentation process, including the use of alternative substrates, defined starter cultures and the modification of fermentation parameters, with the aim of producing an innovative beverage that is improved in terms of its physiochemical, sensory and bioactive properties. The global kombucha market is rapidly increasing, with the rising popularity of the tea attributed in part to its purported health benefits, despite the lack of research in human subjects to substantiate such claims. Accordingly, the incidence of kombucha home-brewing has increased, meaning there is a requirement for individuals to recognise the potential hazards associated with fermentation and the relevant preventative measures to be undertaken to ensure the safe preparation of kombucha. The aim of this review is to provide an update regarding the current knowledge of kombucha production, microbiology, safety and marketing. Full article

We first sought to evaluate the effect of dietary supplementation with the docosahexaenoic acid (DHA)-rich microalgae, Aurantiochytrium limacinum (AURA), on rumen fermentation and the resistance of DHA to degradation and biohydrogenation by rumen microbes through ex vivo fermentation experiments. Subsequently, we sought to quantify the diet-derived DHA content of milk and the impact of AURA on microbial composition and metabolism in a pilot feeding trial with rumen-cannulated dairy cows. To achieve our aims, rumen fluid from cannulated cows was used as inoculum, and the effect of AURA inclusion on fermentation ex vivo was examined. At doses corresponding to the amount of AURA recommended for commercial production animals, only ~10% of DHA was degraded or biohydrogenated by rumen microorganisms. The results show that feeding with AURA had no effect on either total bacterial density or short-chain fatty acid production. Real-time quantitative PCR analysis of the rumen fluid samples collected during a seven-week in vivo trial revealed that microbes related to lactic acid metabolism and methanogenesis were significantly suppressed by the AURA-supplemented diet. The DHA concentration in milk increased over 25-fold with the AURA-supplemented diet and dropped by 30–40% within one week of washout. The addition of A. limacinum biomass to dairy cow diets resulted in positive effects on rumen microbial composition with no adverse effect on fermentation activity. AURA-derived DHA was stable, with only modest degradation in the rumen, and was successfully deposited in milk. This is the first study to investigate the effect of supplementing the diet of dairy cows with a protist-based biomass, namely, on important rumen fermentation parameters and on DHA deposition in milk, using a combination of ex vivo and in vivo approaches. Full article

The microbiome revolution brought the realization that diet, health, and safety for humans in reality means diet, health, and safety for the human holobiont/superorganism. Eating healthier means much more than just feeding human cells. Our diet must also nourish the combination of our microbiome and our connected physiological systems (e.g., the microimmunosome). For this reason, there has been an interest in returning to ancestral “complete” unprocessed foods enriched in microbes, including raw milks. To contribute to this inevitable “nourishing the holobiont” trend, we introduce a systematic risk–benefit analysis tool (evidence mapping), which facilitates transdisciplinary state-of-the-science decisions that transcend single scientific disciplines. Our prior paper developed an evidence map (a type of risk–benefit mind map) for raw vs. processed/pasteurized human breast milk. In the present paper, we follow with a comprehensive evidence map and narrative for raw/natural vs. processed/pasteurized cow’s milk. Importantly, the evidence maps incorporate clinical data for both infectious and non-communicable diseases and allow the impact of modern agricultural, food management, and medical and veterinary monitoring outcomes to be captured. Additionally, we focus on the impact of raw milks (as “complete” foods) on the microimmunosome, the microbiome-systems biology unit that significantly determines risk of the world’s number one cause of human death, non-communicable diseases. Full article

Foodborne illness due to the consumption of contaminated products continues to be a serious public health issue. Bacteriophages might provide a natural and effective way to control and reduce the pathogenic bacterial population on food products. Researchers have conducted various experiments to prove their effectiveness against different pathogens and their ability to act as a natural intervention to control pathogen populations, especially in the food industry. In this study, a cocktail of bacteriophages (phages) was added to wash water in the presence of a high organic load along with commercially used sanitizers (chlorine or Sanidate 5.0) to study the efficacy of the phage–sanitizer combination in the challenge water. It was determined that in the absence of organic loads, the sanitizer by itself or the combination with phages significantly (p < 0.001) reduced the contamination by 3.00–5.00 log CFU/mL. In the presence of organic loads, the sanitizer by itself did not contribute to a significant reduction (p > 0.05) compared to the control. However, the sanitizer–phage combination led to a 3.00-log and 6.00-log reduction (p < 0.001) of the pathogen at the end of 3 and 6 h, respectively, in the presence of high organic loads. Therefore, utilizing a combination treatment (phage–sanitizer) might be one solution to reduce pathogen contamination in the food industry, especially the fresh produce industry, thus providing safe food for consumption. Full article

Dry-cured ham can be contaminated with Listeria monocytogenes during its industrial processing. The use of bacteriocins could ensure the safety of such meat products, but their effect on pathogen physiology is unknown. Therefore, the impact of enterocins A and B on the L. monocytogenes population, and the expression patterns of five genes (inlA, inlB, clpC, fbpA and prfA) related to adhesion/invasion and virulence regulation have been monitored in sliced dry-cured ham during 30 d of storage in refrigeration (4 °C) and temperature-abuse conditions (20 °C). L. monocytogenes strains S2 (serotype 1/2a) and S7-2 (serotype 4b) counts were reduced by 0.5 and 0.6 log units immediately after the application of enterocins A and B, a decrease lower than previously reported. Differences in gene expression were found between the two strains. For strain S2, expression tended to increase for almost all genes up to day seven of storage, whereas this increase was observed immediately after application for strain S7-2; however, overall gene expression was repressed from day one onwards, mainly under temperature-abuse conditions. L. monocytogenes strains investigated in the present work exhibited a mild sensitivity to enterocins A and B in sliced dry-cured ham. Bacteriocins caused changes in the expression patterns of virulence genes associated with adhesion and invasion, although the potential virulence of surviving cells was not enhanced. Full article