Marina Basaglia

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum.

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.

Polyhydroxyalkanoates production by engineered Cupriavidus necator from waste material containing lactose

Cupriavidus necator DSM 545 is a well-known polyhydroxyalkanoates (PHAs) producer, but unable to grow on lactose. The aim of this study was to construct a recombinant strain of C. necator that can use lactose-containing waste material such as cheese whey, to produce PHAs. One of the intracellular PHA depolymerases (phaZ1) of C. necator was chosen to insert the lacZ, lacI and lacO genes of Escherichia coli. This would have the effect to allow polymer production on lactose and, at the same time, to remove part of the PHA intracellular degradation system. Disruption of phaZ1 was achieved by gene replacement after isolating a fragment of this gene and interrupting it with a cartridge containing the lac genes and a synthetic promoter. Growth and polymer production studies of the genetically modified (GM) strain mRePT in lactose, whey permeate and hydrolyzed whey permeate as carbon sources, were performed. Lower PHA degradation and higher yields were obtained compared to the wild-type strain. Inactivation of the putative depolymerase gene phaZ3 on mRePT recombinant strain was also reported.

Dimethyl carbonate and switchable anionic surfactants: two effective tools for the extraction of polyhydroxyalkanoates from microbial biomass

The availability of green and cheap technologies to recover polyhydroxyalkanoates (PHAs) from microbial biomass is crucial for the development of a reliable and sustainable production chain. Here, two novel protocols are proposed to extract PHAs from Cupriavidus necator. The first method is based on PHA-extraction with dimethyl carbonate (DMC), a green solvent that is completely biodegradable and less harmful to humans and the environment than most solvents. The procedure can be applied directly to concentrated microbial slurries or to dry biomass, affording very high polymer recovery (>85%) and excellent purity (>95%). No degradation/decomposition of the polymer is observed in both cases. The second protocol uses fatty acid carboxylates as surfactants, which disrupt cell membranes, providing excellent polymer recovery (>99%) and high purity (>90%). Ammonium laurate can be successfully used and easily recycled (98%) by lowering the pH through CO2 addition. Therefore, both protocols reported here are effective and sustainable: the recovery and purity of the obtained PHAs are very high, the use of toxic chemicals is avoided, and the recycling of various solvents/surfactants used in the processes is optimal.

Resuscitation of Viable But Not Culturable Sinorhizobium meliloti 41 pRP4-luc: Effects of Oxygen and Host Plant

A plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and stably inherited in Sinorhizobium meliloti 41 as a reporter gene. The strain obtained, S. meliloti 41/pRP4-luc, and its parental strain served as a model system for viable but not culturable (VBNC) resuscitation experiments in both in vitro and soil samples. Incubation under oxygen (O2) concentrations varying from 1% to atmospheric levels did not result in resuscitation. A demonstration of recovery was attained through exposure to the appropriate concentrations of antibiotics, bacteriostatic chloramphenicol, and bactericidal ampicillin. The resuscitation ratio was 1 recovered VBNC cell in every 105 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC+) bacteria. Although isolated VBNC rhizobia were unable to nodulate Medicago sativa, which apparently did not enhance VBNC reversion, resuscitated bacteria maintained their symbiotic properties. Soil experiments showed that the lack of O2 leads to onset of VBNC status as in liquid microcosm, but the number of recoverable and culturable cells decreased more drastically in soil.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases

The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch‐based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon‐optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α‐amylase (SFA1) genes were δ‐integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1‐SFA1] and MEL2[TLG1‐SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch‐to‐ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre‐treatment or commercial enzyme addition) using industrial yeast strains co‐secreting both a glucoamylase and α‐amylase. Biotechnol. Bioeng. 2015;112: 1751–1760.

Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium

The production of bacteriocins from cheap substrates could be useful for many food industrial applications. This study aimed at determining the conditions needed for optimal production of enterocins SD1, SD2, SD3 and SD4 secreted by Enterococcus faecium strains SD1, SD2, SD3 and SD4, respectively. To our knowledge, this is the first use of cheese whey—a low-cost milk by-product—as a substrate for bacteriocin production by E. faecium; skimmed milk and MRS broths were used as reference media. This cheese manufacturing residue proved to be a promising substrate for the production of bacteriocins. However, the levels of secreted antimicrobial compounds were lower than those achieved by E. faecium strains in MRS broth. Bacteriocin production was affected strongly by physical and chemical factors such as growth temperature, time of incubation, pH, and the chemical composition of the culture medium. The optimal temperature and time of incubation supporting the highest bacteriocin production was determined for each strain. Different types, sources and amounts of organic nitrogen, sugar, and inorganic salts played an essential role in bacteriocin secretion. E. faecium strains SD1 and SD2—producing high bacteriocin levels both in cheese whey and skimmed milk—could be of great interest for potential applications in cheese-making.

Field release of genetically marked Azospirillum brasilense in association with Sorghum bicolor L.

The agronomic impact of genetically tagged azospirilla (Azospirillum brasilense)was assessed in open field and their fluctuation were monitored in the soil/rhizosphere. Strain performance, upon inoculation of sorghum, was evaluated over a two-years period; agronomic treatments included nitrogen application (0, 80, 160 kg ha−1), and types of inoculant (Sp245 lacZ, Sp6 gusA, Sp6 IAA++gusA). Grain yield was higher for inoculated seed plots than in non-inoculated ones, whereas nitrogen content, biomass of plant residues and nitrogen in plant residues gave values that were not statistically different. Root length density (RLD) of sorghum at the end of the stem elongation stage was affected only by the indole-3-acetic acid (IAA) overproducer Azospirillum strain (A. brasilense Sp6 IAA++gusA) with respect to the normal IAA producer (A. brasilense Sp6 gusA), being higher in the first 40 cm of depth, notwithstanding the level of nitrogen fertilization. The traceability of the released genetically modified strains enabled to monitor their ability to colonise soil and roots. Moreover, the genetic modification per se vs. the non-modified counterpart, did not affect the culturable aerobic population in soil, microfungi, streptomycetes, fluorescent pseudomonads, soil microbial biomass, or some microbial activities, all selected as important indicators.

Designing industrial yeasts for the consolidated bioprocessing of starchy biomass to ethanol.

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one step process, is a promising strategy for the effective ethanol production from cheap lignocellulosic and starchy materials. CBP requires a highly engineered microbial strain able to both hydrolyze biomass with enzymes produced on its own and convert the resulting simple sugars into high-titer ethanol. Recently, heterologous production of cellulose and starch-degrading enzymes has been achieved in yeast hosts, which has realized direct processing of biomass to ethanol. However, essentially all efforts aimed at the efficient heterologous expression of saccharolytic enzymes in yeast have involved laboratory strains and much of this work has to be transferred to industrial yeasts that provide the fermentation capacity and robustness desired for large scale bioethanol production. Specifically, the development of an industrial CBP amylolytic yeast would allow the one-step processing of low-cost starchy substrates into ethanol. This article gives insight in the current knowledge and achievements on bioethanol production from starchy materials with industrial engineered S. cerevisiae strains.

Effects of heat treatment on microbial communities of granular sludge for biological hydrogen production.

Dark fermentation shares many features with anaerobic digestion with the exception that to maximize hydrogen production, methanogens and hydrogen-consuming bacteria should be inhibited. Heat treatment is widely applied as an inoculum pre-treatment due to its effectiveness in inhibiting methanogenic microflora but it may not exclusively select for hydrogen-producing bacteria. This work evaluated the effects of heat treatment on microbial viability and structure of anaerobic granular sludge. Heat treatment was carried out on granular sludge at 100 °C with four residence times (0.5, 1, 2 and 4 h). Hydrogen production of treated sludges was studied from glucose by means of batch test at different pH values. Results indicated that each heat treatment strongly influenced the granular sludge resulting in microbial communities having different hydrogen productions. The highest hydrogen yields (2.14 moles of hydrogen per mole of glucose) were obtained at pH 5.5 using the sludge treated for 4 h characterized by the lowest CFU concentration (2.3 × 10(3)CFU/g sludge). This study demonstrated that heat treatment should be carefully defined according to the structure of the sludge microbial community, allowing the selection of highly efficient hydrogen-producing microbes.

An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic yellow vein virus, by means of seed inoculants

Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudomonas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYYV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the colonization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5 × 106 CFU g−1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations.