Exploiting the microbiome associated with normal and abnormal sprouting rice (Oryza sativa L.) seed phenotypes through a metabarcoding approach.

Rice germination and seedlings' growth are crucial stages that influence crop establishment and productivity. These performances depend on several factors, including the abundance and diversity of seed microbial endophytes. Two popular rainfed rice varieties cultivated in Cameroon, NERICA 3 and NERICA 8, were used for investigating the seed-associated microbiome using the Illumina-based 16 S rRNA gene. Significant differences were observed in terms of richness index between normal and abnormal seedlings developed from sprouting seeds, although no significant species evenness index was assessed within either phenotype. Two hundred ninety-two bacterial amplicon sequence variants were identified in seed microbiome of the rice varieties, and principal coordinate analysis revealed that microbial communities formed two distinct clusters in normal and abnormal seedling phenotypes. Overall, 38 bacteria genera were identified, belonging to 6 main phyla. Furthermore, the core microbiome was defined, and the differential abundance of 28 bacteria genera was assessed. Based on the collected results, putative bacterial genera were directly correlated with the development of normal seedlings. For most genera that are recognised to include beneficial species, such as Brevundimonas, Sphingomonas, Exiguobacterium, Luteibacter, Microbacterium and Streptomyces, a significant increase of their relative abundance was found in normal seedlings. Additionally, in abnormal seedlings, we also observed an increased abundance of the genera Kosakonia and Paenibacillus, which might have controversial aspects (beneficial or pathogenic), together with the presence of some genera (Clostridium sensu stricto) that are commonly correlated to sick plants. The putative functional gene annotation revealed the higher abundance of genes related to the metabolic biosynthesis of soluble carbohydrates and starch, tryptophan, nucleotides and ABC transporters in normal seedlings. Data presented in this study may help in further understanding the importance of the seed endophyte microbiome for driving a correct development of rice plants at the early stages and to identify possible beneficial bacteria for technological applications aimed to increase seed quality and crop productivity.

A Microbial Co-Culturing System for Producing Cellulose-Hyaluronic Acid Composites.

In this study, a co-culture system combining bacterial cellulose (BC) producers and hyaluronic acid (HA) producers was developed for four different combinations. AAB of the genus Komagataeibacter sp. and LAB of the Lactocaseibacillus genus were used to produce BC and HA, respectively. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate changes in BC-HA composites chemical and morphological structure. Water absorption, uptake, and antibacterial properties were also tested. Outcomes highlighted a higher bacterial cellulose yield and the incorporation of hyaluronic acid into the composite. The presence of hyaluronic acid increased fiber dimension-nearly doubled for some combinations-which led to a decreased crystallinity of the composites. Different results were observed based on the BC producer and HA producer combination. However, water holding capacity (WHC) in all the samples improved with the presence of HA, while water uptake worsened. A thymol-enriched BC-HA composite showed high antibacterial activity against Escherichia coli DSM 30083T and Staphylococcus aureus DSM 20231T. Results could contribute to opening new applications in the cosmetics or pharmaceutical fields.

Acetic acid bacteria in agro-wastes: from cheese whey and olive mill wastewater to cellulose.

In this study, cheese whey and olive mill wastewater were investigated as potential feedstocks for producing bacterial cellulose by using acetic acid bacteria strains. Organic acids and phenolic compounds composition were assayed by high-pressure liquid chromatography. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate modifications in bacterial cellulose chemical and morphological structure. Cheese whey was the most efficient feedstock in terms of bacterial cellulose yield (0.300 g of bacterial cellulose/gram of carbon source consumed). Bacterial cellulose produced in olive mill wastewater presented a more well-defined network compared to pellicles produced in cheese whey, resulting in a smaller fiber diameter in most cases. The analysis of bacterial cellulose chemical structure highlighted the presence of different chemical bonds likely to be caused by the adsorption of olive mill wastewater and cheese whey components. The crystallinity ranged from 45.72 to 80.82%. The acetic acid bacteria strains used in this study were characterized by 16S rRNA gene sequencing, allowing to assign them to Komagataeibacter xylinus and Komagataeibacter rhaeticus species. This study proves the suitability to perform sustainable bioprocesses for producing bacterial cellulose, combining the valorisation of agro-wastes with microbial conversions carried out by acetic acid bacteria. The high versatility in terms of yield, morphology, and fiber diameters obtained in cheese whey and olive mill wastewater contribute to set up fundamental criteria for developing customized bioprocesses depending on the final use of the bacterial cellulose. KEY POINTS: • Cheese whey and olive mill wastewater can be used for bacterial cellulose production. • Bacterial cellulose structure is dependent on the culture medium. • Komagataeibacter strains support the agro-waste conversion in bacterial cellulose.

Defining Paenibacillus azoreducens (P8) and Acetobacter pasteurianus (UMCC 2951) strains performances in producing acetic acid.

In this study, spore-forming bacteria isolated from saccharified rice were selected for producing acetic acid. From the screening of 15 strains, P8 strain was chosen as a candidate. The strain was identified as Paenibacillus azoreducens by 16S rRNA analysis (99.85% similarity with P. azoreducens CM1T). Acetic acid is the main component of vinegar but also an industrial commodity produced by chemical synthesis. Sustainable routes for obtaining acetic acid are of great interest for decreasing the environmental impact generated by chemical syntheses. Biological acetic acid production is effective for vinegar production by acetic acid bacteria, but it cannot economically compete with the chemical synthesis for producing it as a pure commodity. Considering the need to improve the yield of pure acetic acid produced by microbial conversions, in this study, P8 strain was chosen for designing processes in different fermentation conditions. Tests were conducted in single and semi-continuous systems, using rice wine as substrate. Acetic acid produced by P8 strain was compared with that of Acetobacter pasteurianus (UMCC 2951), a strain known for producing acetic acid from rice wine. Even though the fermentation performances of P. azoreducens P8 were slightly lower than those of acetic acid bacteria usually used for vinegar production, results highlight its suitability for producing acetic acid. The final acetic acid produced by P. azoreducens P8 was 73 g/L, in a single stage fermentation, without losses. In nine cycles of semi-continuous regime the average of acetification rate was 0.814 (g/L/days). Two main attributes of P. azoreducens P8 are of relevance for producing acetic acid, namely the ability to grow at temperature higher (+ 37°C), than mesophilic acetic acid bacteria, and the absence of cytoplasmic assimilation of acetic acid. These features allow to design multiple strains cultures, in which P. azoreducens can acts as a helper strain. Based on our results, the new isolate P. azoreducens P8 can be propagated in fermenting broths for boosting acetic acid production, under the selected conditions, and used in combination with acetic acid bacteria to produce biological acetic acid, as a non-food grade commodity.

Better under stress: Improving bacterial cellulose production by Komagataeibacter xylinus K2G30 (UMCC 2756) using adaptive laboratory evolution.

Among naturally produced polymers, bacterial cellulose is receiving enormous attention due to remarkable properties, making it suitable for a wide range of industrial applications. However, the low yield, the instability of microbial strains and the limited knowledge of the mechanisms regulating the metabolism of producer strains, limit the large-scale production of bacterial cellulose. In this study, Komagataeibacter xylinus K2G30 was adapted in mannitol based medium, a carbon source that is also available in agri-food wastes. K. xylinus K2G30 was continuously cultured by replacing glucose with mannitol (2% w/v) for 210 days. After a starting lag-phase, in which no changes were observed in the utilization of mannitol and in bacterial cellulose production (cycles 1-25), a constant improvement of the phenotypic performances was observed from cycle 26 to cycle 30, accompanied by an increase in mannitol consumption. At cycle 30, the end-point of the experiment, bacterial cellulose yield increased by 38% in comparision compared to cycle 1. Furthermore, considering the mannitol metabolic pathway, D-fructose is an intermediate in the bioconversion of mannitol to glucose. Based on this consideration, K. xylinus K2G30 was tested in fructose-based medium, obtaining the same trend of bacterial cellulose production observed in mannitol medium. The adaptive laboratory evolution approach used in this study was suitable for the phenotypic improvement of K. xylinus K2G30 in bacterial cellulose production. Metabolic versatility of the strain was confirmed by the increase in bacterial cellulose production from D-fructose-based medium. Moreover, the adaptation on mannitol did not occur at the expense of glucose, confirming the versatility of K2G30 in producing bacterial cellulose from different carbon sources. Results of this study contribute to the knowledge for designing new strategies, as an alternative to the genetic engineering approach, for bacterial cellulose production.

Candidate Acetic Acid Bacteria Strains for Levan Production.

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099 T, Kozakia baliensis DSM 14400 T and Gluconobacter cerinus DSM 9533 T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099 T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400 T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

Assessing effectiveness of Komagataeibacter strains for producing surface-microstructured cellulose via guided assembly-based biolithography.

In this study, a medical device made of surface microstructured bacterial cellulose was produced using cellulose-producing acetic acid bacteria wild-type strains in combination with guided assembly-based biolithography. The medical device aims at interfering with the cell's focal adhesion establishment and maturation around implantable devices placed in soft tissues by the symmetrical array on its surface. A total of 25 Komagataeibacter strains was evaluated over a three-step selection. In the first step, the ability of strains to produce a suitable bacterial cellulose layer with high production yield was examined, then nine strains, with a uniform and smooth layer of bacterial cellulose, were cultured in a custom-made silicone bioreactor and finally the characteristics of the symmetrical array of topographic features on the surface were analysed. Selected strains showed high inter and intra species variability in bacterial cellulose production. The devices obtained by K2G30, K1G4, DSM 46590 (Komagataeibacter xylinus), K2A8 (Komagataeibacter sp.) and DSM 15973T (Komagataeibacter sucrofermentas) strains were pouched-formed with hexagonal surface pattern required for reducing the formation of fibrotic tissue around devices, once they are implanted in soft tissues. Our findings revealed the effectiveness of the selected Komagataeibacter wild-type strains in producing surface microstructured bacterial cellulose pouches for making biomedical devices.

Tannery Wastewater Recalcitrant Compounds Foster the Selection of Fungi in Non-Sterile Conditions: A Pilot Scale Long-Term Test.

This study demonstrated that a microbial community dominated by fungi can be selected and maintained in the long-term under non-sterile conditions, in a pilot-scale packed-bed reactor fed with tannery wastewater. During the start-up phase, the reactor, filled with 0.6 m3 of polyurethane foam cubes, was inoculated with a pure culture of Aspergillus tubingensis and Quebracho tannin, a recalcitrant compound widely used by tannery industry, was used as sole carbon source in the feeding. During the start-up, fungi grew attached as biofilm in carriers that filled the packed-bed reactor. Subsequently, the reactor was tested for the removal of chemical oxygen demand (COD) from an exhaust tanning bath collected from tanneries. The entire experiment lasted 121 days and average removals of 29% and 23% of COD and dissolved organic carbon (DOC) from the tannins bath were achieved, respectively. The evolution of the microbial consortium (bacteria and fungi) was described through biomolecular analyses along the experiment and also developed as a function of the size of the support media.

A New Ciboria sp. for Soil Mycoremediation and the Bacterial Contribution to the Depletion of Total Petroleum Hydrocarbons.

A Ciboria sp. strain (Phylum Ascomycota) was isolated from hydrocarbon-polluted soil of an abandoned oil refinery in Italy. The strain was able to utilize diesel oil as a sole carbon source for growth. Laboratory-scale experiments were designed to evaluate the use of this fungal strain for treatment of the polluted soil. The concentration of total petroleum hydrocarbons (TPH) in the soil was 8,538 mg/kg. Mesocosms containing the contaminated soil were inoculated with the fungal strain at 1 or 7%, on a fresh weight base ratio. After 90 days of incubation, the depletion of TPH contamination was of 78% with the 1% inoculant, and 99% with the 7% inoculant. 16S rDNA and ITS metabarcoding of the bacterial and fungal communities was performed in order to evaluate the potential synergism between fungi and bacteria in the bioremediation process. The functional metagenomic prediction indicated Arthrobacter, Dietzia, Brachybacerium, Brevibacterium, Gordonia, Leucobacter, Lysobacter, and Agrobacterium spp. as generalist saprophytes, essential for the onset of hydrocarbonoclastic specialist bacterial species, identified as Streptomyces, Nocardoides, Pseudonocardia, Solirubrobacter, Parvibaculum, Rhodanobacter, Luteiomonas, Planomicrobium, and Bacillus spp., involved in the TPH depletion. The fungal metabolism accelerated the onset of specialist over generalist bacteria. The capacity of the Ciboria sp. to deplete TPH in the soil in treatment was also ascertained.

Yeasts and Lactic Acid Bacteria for Panettone Production: An Assessment of Candidate Strains.

The recovery of yeasts and lactic acid bacteria (LAB) involved in sourdough fermentation is the first step in the selection of starters with suitable technological aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (MA and MB) and the derived doughs (samples A and B) were collected from a bakery during artisanal Panettone manufacture. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 77 isolates were obtained and characterized. Representative strains of yeasts and LAB were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. Moreover, the volatile organic compounds (VOCs) produced in the collected samples were detected and correlated to the species found in the same samples. The results highlighted the occurrence of Kazachstania humilis in both samples A and B, while Saccharomyces cerevisiae strains were detected only in samples B. Among LAB, Fructilactobacillus sanfranciscensis was the main species detected in both sourdoughs. Furthermore, strains belonging to the species Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, and Leuconostoc mesenteroides were assessed in the dough samples.

Degradation of BTEX mixture by a new Pseudomonas putida strain: role of the quorum sensing in the modulation of the upper BTEX oxidative pathway.

A new Pseudomonas putida strain (AQ8) was isolated from a decommissioned oil refinery's soil in Italy and characterized for its ability to degrade BTEX. The draft genome of the new strain was sequenced and annotated for genes that encode enzymes putatively involved in BTEX degradation and quorum sensing. The strain was transformed with a plasmid expressing lactonase, which cleaves the autoinducer quorum sensing signal molecule, the acyl-homoserine lactone, to obtain a quorum sensing minus strain. P. putida AQ8 depleted the 40% on average of all the components of the initial BTEX concentration in 36 h. The quorum sensing minus strain, in the same time interval, depleted only the 10% of the initial BTEX concentration. The role of quorum sensing in regulating the expression of the annotated benzene/toluene dioxygenase gene (benzA) and biphenyl/toluene/benzene dioxygenase (bphA) genes, which are involved in BTEX degradation, was studied by quantitative RT-real-time quantitative (q)PCR analysis. The qPCR data showed decreased levels of expression of the benzA and bphA genes in the quorum sensing minus strain. Our results showed, for the first time, quorum sensing modulation of the level of transcription of dioxygenase genes in the upper BTEX oxidation pathway.

Genome sequencing and phylogenetic analysis of K1G4: a new Komagataeibacter strain producing bacterial cellulose from different carbon sources.

OBJECTIVE: The objective of this study was to evaluate the ability of a new Komagataeibacter xylinus strain in producing bacterial cellulose from glucose, mannitol and glycerol, and to assess the genome sequencing with special focus on bacterial cellulose related genes. RESULTS: Bacterial cellulose production during 9 days of cultivation was tested in glucose, mannitol and glycerol, respectively. Differences in the bacterial cellulose kinetic formation was observed, with a final yield of 9.47 g/L in mannitol, 8.30 g/L in glycerol and 7.57 g/L in glucose, respectively. The draft genome sequencing of K1G4 was produced, revealing a genome of 3.09 Mbp. Two structurally completed cellulose synthase operons and a third copy of the catalytic subunit of cellulose synthase were found. By using phylogenetic analysis, on the entire rRNA operon sequence, K1G4 was found to be closely related to Komagataeibacter xylinus LMG 1515T and K. xylinus K2G30. CONCLUSIONS: The different yields of bacterial cellulose produced on glucose, mannitol and glycerol can be correlated with the third copy of bcsAB operon harboured by K1G4, making it a versatile strain for industrial applications.

Exploring K2G30 Genome: A High Bacterial Cellulose Producing Strain in Glucose and Mannitol Based Media.

Demands for renewable and sustainable biopolymers have rapidly increased in the last decades along with environmental issues. In this context, bacterial cellulose, as renewable and biodegradable biopolymer has received considerable attention. Particularly, acetic acid bacteria of the Komagataeibacter xylinus species can produce bacterial cellulose from several carbon sources. To fully exploit metabolic potential of cellulose producing acetic acid bacteria, an understanding of the ability of producing bacterial cellulose from different carbon sources and the characterization of the genes involved in the synthesis is required. Here, K2G30 (UMCC 2756) was studied with respect to bacterial cellulose production in mannitol, xylitol and glucose media. Moreover, the draft genome sequence with a focus on cellulose related genes was produced. A pH reduction and gluconic acid formation was observed in glucose medium which allowed to produce 6.14 ± 0.02 g/L of bacterial cellulose; the highest bacterial cellulose production obtained was in 1.5% (w/v) mannitol medium (8.77 ± 0.04 g/L), while xylitol provided the lowest (1.35 ± 0.05 g/L) yield. Genomic analysis of K2G30 revealed a peculiar gene sets of cellulose synthase; three bcs operons and a fourth copy of bcsAB gene, that encodes the catalytic core of cellulose synthase. These features can explain the high amount of bacterial cellulose produced by K2G30 strain. Results of this study provide valuable information to industrially exploit acetic acid bacteria in producing bacterial cellulose from different carbon sources including vegetable waste feedstocks containing mannitol.

Hydrocarbonoclastic Ascomycetes to enhance co-composting of total petroleum hydrocarbon (TPH) contaminated dredged sediments and lignocellulosic matrices.

Four new Ascomycete fungi capable of degrading diesel oil were isolated from sediments of a river estuary mainly contaminated by shipyard fuels or diesel oil. The isolates were identified as species of Lambertella, Penicillium, Clonostachys, and Mucor. The fungal candidates degraded and adsorbed the diesel oil in suspension cultures. The Lambertella sp. isolate displayed the highest percentages of oxidation of diesel oil and was characterised by the capacity to utilise the latter as a sole carbon source. This isolate showed extracellular laccase and Mn-peroxidase activities in the presence of diesel oil. It was tested for capacity to accelerate the process of decontamination of total petroleum hydrocarbon contaminated sediments, co-composted with lignocellulosic residues and was able to promote the degradation of 47.6% of the TPH contamination (54,074 ± 321 mg TPH/Kg of sediment) after two months of incubation. The response of the bacterial community during the degradation process was analysed by 16S rRNA gene meta-barcoding.

Oxidative fermentations and exopolysaccharides production by acetic acid bacteria: a mini review.

Acetic acid bacteria are versatile organisms converting a number of carbon sources into biomolecules of industrial interest. Such properties, together with the need to limit chemical syntheses in favor of more sustainable biological processes, make acetic acid bacteria appropriate organisms for food, chemical, medical, pharmaceutical and engineering applications. At current, well-established bioprocesses by acetic acid bacteria are those derived from the oxidative pathways that lead to organic acids, ketones and sugar derivates. Whereas emerging applications include biopolymers, such as bacterial cellulose and fructans, which are getting an increasing interest for the biotechnological industry. However, considering the industrial demand of high performing bioprocesses, the production yield of metabolites obtained by acetic acid bacteria, is still not satisfying. In this paper we review the major acetic acid bacteria industrial applications, considering the current status of bioprocesses. We will also describe new biotechnological advances in order to optimize the industrial production, offering also an overview on future directions.

Biotechnological production of cellulose by acetic acid bacteria: current state and perspectives.

Bacterial cellulose is an attractive biopolymer for a number of applications including food, biomedical, cosmetics, and engineering fields. In addition to renewability and biodegradability, its unique structure and properties such as chemical purity, nanoscale fibrous 3D network, high water-holding capacity, high degree of polymerization, high crystallinity index, light transparency, biocompatibility, and mechanical features offer several advantages when it is used as native polymer or in composite materials. Structure and properties play a functional role in both the biofilm life cycle and biotechnological applications. Among all the cellulose-producing bacteria, acetic acid bacteria of the Komagataeibacter xylinus species play the most important role because they are considered the highest producers. Bacterial cellulose from acetic acid bacteria is widely investigated as native and modified biopolymer in functionalized materials, as well as in terms of differences arising from the static or submerged production system. In this paper, the huge amount of knowledge on basic and applied aspects of bacterial cellulose is reviewed to the aim to provide a comprehensive viewpoint on the intriguing interplay between the biological machinery of synthesis, the native structure, and the factors determining its nanostructure and applications. Since in acetic acid bacteria biofilm and cellulose production are two main phenotypes with industrial impact, new insights into biofilm production are provided.