Current status and future trends of microbial and nematode-based biopesticides for biocontrol of crop pathogens.

The increasing public demand to avoid the use of synthetic pesticides and fertilizers in agricultural production systems, causing serious environmental damages, has challenged industry to develop new and effective solutions to manage and control phytopathogens. Biopesticides, particularly microbial-based biopesticides, are a promising new alternative with high biodegradability, specificity, suitability for incorporation into integrated pest management practices, low likelihood of resistance development, and practically no known human health risks. However: expensive production methods, narrow action spectra, susceptibility to environmental conditions, short shelf life, poor storage stability, legislation registry constraints, and general lack of knowledge are slowing down their adoption. In addition to regulatory framework revisions and improved training initiatives, improved preservation methods, thoughtfully designed formulations, and field test validations are needed to offer new microbial- and nematode-based biopesticides with improved efficacy and increased shelf-life. During the last several years, substantial advancements in biopesticide production have been developed. The novelty part of this review written in 2023 is to summarize (i) mechanisms of action of beneficial microorganisms used to increase crop performance and (ii) successful formulation including commercial products for the biological control of phytopathogens based on microorganisms, nematode and/or metabolites.

Changes in chemical properties and microbial communities' composition of a forest litter-based biofertilizer produced through aerated solid-state culture under different oxygen conditions.

Fermented forest litter (FFL) is a bioproduct used as biofertilizer for several decades in Eastern Asia and Latin America. It is locally handcrafted by farmers in anaerobic conditions by fermenting forest litter added with agricultural by-products such as whey, cereal bran, and molasses. The aim of this study was to characterize the FFL process and product through gas and liquid chromatography analyses. It also provides some highlights on the influence of O2 on this solid-state culture. Under anoxic condition, a maximum CO2 production rate (CDPR) of 0.41 mL/h∙g dry matter (dm) was reached after 8 days. The main volatile organic compounds (VOCs) were ethanol and ethyl acetate, with a production rate profile similar to CDPR. After 21 days of culture, no residual sucrose nor lactose was detected. Lactic and acetic acids reached 58.8 mg/g dm and 10.2 mg/g dm, respectively, ensuring the acidification of the matrix to a final pH of 4.72. A metabarcoding analysis revealed that heterolactic acid bacteria (Lentilactobacillus, Leuconostoc), homolactic acid bacteria (Lactococcus), and yeasts (Saccharomyces, Clavispora) were predominant. Predicted genes in the microbiome confirmed the potential link between detected bacteria and acids and VOCs produced. When O2 was fed to the cultures, final pH reached values up to 8.5. No significant amounts of lactic nor acetic acid were found. In addition, a strong shift in microbial communities was observed, with a predominance of Proteobacteria and molds, among which are potential pathogens like Fusarium species. This suggests that particular care must be brought to maintain anoxic conditions throughout the process.

Chemical and Microbial Characterization of Fermented Forest Litters Used as Biofertilizers.

The excessive use of chemicals in intensive agriculture has had a negative impact on soil diversity and fertility. A strategy for developing sustainable agriculture could rely on the use of microbial-based fertilizers, known as biofertilizers. An alternative to marketed products could be offered to small farmers if they could produce their own biofertilizers using forest litters, which harbor one of the highest microbial diversities. The aim of this study is to characterize microbial communities of Fermented Forest Litters (FFL), assuming that the fermentation process will change both their abundance and diversity. We investigated two types of differing in the chemical composition of the initial litters used and the climatic context of the forest where they are originated from. The abundance and diversity of bacterial and fungal communities were assessed using quantitative PCR and molecular genotyping techniques. The litter chemical compositions were compared before and after fermentation using Infrared spectrometry. Results obtained showed that fermentation increased the abundance of bacteria but decreased that of fungi. Low pH and change in organic matter composition observed after fermentation also significantly reduced the α-diversity of both bacterial and fungal communities. The higher proportion of aliphatic molecules and lower C/N of the FFLs compared to initial litters indicate that FFLs should be rapidly decomposed once added into the soil. This preliminary study suggests that the agronomic interest of FFLs used as biofertilizers is probably more related to the contribution of nutrients easily assimilated by plants than to the diversity of microorganisms that compose it. Further studies must be conducted with sequencing techniques to identify precisely the microbial species likely to be beneficial to plant growth.

Phenol biodegradation by the thermoacidophilic archaeon Sulfolobus solfataricus 98/2 in a fed-batch bioreactor.

Toxic at low concentrations, phenol is one of the most common organic pollutants in air and water. In this work, phenol biodegradation was studied in extreme conditions (80°C, pH = 3.2) in a 2.7 l bioreactor with the thermoacidophilic archaeon Sulfolobus solfataricus 98/2. The strain was first acclimatized to phenol on a mixture of glucose (2000 mg l(-1)) and phenol (94 mg l(-1)) at a constant dissolved oxygen concentration of 1.5 mg l(-1). After a short lag-phase, only glucose was consumed. Phenol degradation then began while glucose was still present in the reactor. When glucose was exhausted, phenol was used for respiration and then for biomass build-up. After several batch runs (phenol < 365 mg l(-1)), specific growth rate (µ(X)) was 0.034 ± 0.001 h(-1), specific phenol degradation rate (q(P)) was 57.5 ± 2 mg g(-1) h(-1), biomass yield (Y(X/P)) was 52.2 ± 1.1 g mol(-1), and oxygen yield factor (Y(X/O2)) was 9.2 ± 0.2 g mol(-1). A carbon recovery close to 100% suggested that phenol was exclusively transformed into biomass (35%) and CO(2) (65%). Molar phenol oxidation constant (Y(O2/P)) was calculated from stoichiometry of phenol oxidation and introducing experimental biomass and CO(2) conversion yields on phenol, leading to values varying between 4.78 and 5.22 mol mol(-1). Respiratory quotient was about 0.84 mol mol(-1), very close to theoretical value (0.87 mol mol(-1)). Carbon dioxide production, oxygen demand and redox potential, monitored on-line, were good indicators of growth, substrate consumption and exhaustion, and can therefore be usefully employed for industrial phenol bioremediation in extreme environments.

Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima Part II: modeling and experimental approaches for hydrogen production.

BACKGROUND: Thermotoga maritima is a hyperthermophilic bacterium known to produce hydrogen from a large variety of substrates. The aim of the present study is to propose a mathematical model incorporating kinetics of growth, consumption of substrates, product formations, and inhibition by hydrogen in order to predict hydrogen production depending on defined culture conditions. RESULTS: Our mathematical model, incorporating data concerning growth, substrates, and products, was developed to predict hydrogen production from batch fermentations of the hyperthermophilic bacterium, T. maritima. It includes the inhibition by hydrogen and the liquid-to-gas mass transfer of H2, CO2, and H2S. Most kinetic parameters of the model were obtained from batch experiments without any fitting. The mathematical model is adequate for glucose, yeast extract, and thiosulfate concentrations ranging from 2.5 to 20 mmol/L, 0.2-0.5 g/L, or 0.01-0.06 mmol/L, respectively, corresponding to one of these compounds being the growth-limiting factor of T. maritima. When glucose, yeast extract, and thiosulfate concentrations are all higher than these ranges, the model overestimates all the variables. In the window of the model validity, predictions of the model show that the combination of both variables (increase in limiting factor concentration and in inlet gas stream) leads up to a twofold increase of the maximum H2-specific productivity with the lowest inhibition. CONCLUSIONS: A mathematical model predicting H2 production in T. maritima was successfully designed and confirmed in this study. However, it shows the limit of validity of such mathematical models. Their limit of applicability must take into account the range of validity in which the parameters were established.

Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima part I: effects of sulfured nutriments, with thiosulfate as model, on hydrogen production and growth.

BACKGROUND: Thermotoga maritima and T. neapolitana are hyperthermophile bacteria chosen by many research teams to produce bio-hydrogen because of their potential to ferment a wide variety of sugars with the highest theoretical H2/glucose yields. However, to develop economically sustainable bio-processes, the culture medium formulation remained to be optimized. The main aim of this study was to quantify accurately and specifically the effect of thiosulfate, used as sulfured nutriment model, on T. maritima growth, yields and productivities of hydrogen. The results were obtained from batch cultures, performed into a bioreactor, carefully controlled, and specifically designed to prevent the back-inhibition by hydrogen. RESULTS: Among sulfured nutriments tested, thiosulfate, cysteine, and sulfide were found to be the most efficient to stimulate T. maritima growth and hydrogen production. In particular, under our experimental conditions (glucose 60 mmol L-1 and yeast extract 1 g L-1), the cellular growth was limited by thiosulfate concentrations lower than 0.06 mmol L-1. Under these conditions, the cellular yield on thiosulfate (Y X/Thio) could be determined at 3617 mg mmol-1. In addition, it has been shown that the limitations of T. maritima growth by thiosulfate lead to metabolic stress marked by a significant metabolic shift of glucose towards the production of extracellular polysaccharides (EPS). Finally, it has been estimated that the presence of thiosulfate in the T. maritima culture medium significantly increased the cellular and hydrogen productivities by a factor 6 without detectable sulfide production. CONCLUSIONS: The stimulant effects of thiosulfate at very low concentrations on T. maritima growth have forced us to reconsider its role in this species and more probably also in all thiosulfato-reducer hyperthermophiles. Henceforth, thiosulfate should be considered in T. maritima as (1) an essential sulfur source for cellular materials when it is present at low concentrations (about 0.3 mmol g-1 of cells), and (2) as both sulfur source and detoxifying agent for H2 when thiosulfate is present at higher concentrations and, when, simultaneously, the pH2 is high. Finally, to improve the hydrogen production in bio-processes using Thermotoga species, it should be recommended to incorporate thiosulfate in the culture medium.

Optimisation of a culture medium containing fish silage for L-lysine production by Corynebacterium glutamicum.

In a first step the effects of 10 components of a culture medium designed for L-lysine production were evaluated with a 2(10-6) factorial design. Among them, glucose, fish silage, and ammonium sulphate showed a significant effect. In a second step, an orthogonal-central composite experimental design and response surface methodology was performed with five from the 10 initial compounds. The determination coefficient (R2) of the fitted second-order model was 0.990. L-lysine production with the optimised medium increased 2.6 times as compared with the original medium.

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1)) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

Fruity aroma production by Ceratocystis fimbriata in solid cultures from agro-industrial wastes

Solid state fermentations were carried out to test the efficacy of Ceratocystis fimbriata to grow on different agro-industrial substrates and aroma production. Seven media were prepared using cassava bagasse, apple pomace, amaranth and soya bean. All the media supported fungal growth. While amaranth medium produced pineapple aroma, media containing cassava bagasse, apple pomace and soya bean produced a strong fruity aroma. The aroma production was growth dependent and the maximum aroma intensity was detected a few hours before or after the maximun respirometric activity. Sixteen compounds were separated by gas cromatography of the components present in the headspace and fifteen of them were identified as acid (l), alcohols (6), aldehyde (l), ketones (2) and esters (5).

Coffee residues as substrates for aroma production by Ceratocystis fimbriata in solid state fermentation

Neste trabalho duas diferentes cepas de Ceratocystis fimbriata foram testadas para a producão de aromas frutais em fermentacão no estado sólido (FES) utilizando como substratos casca e polpa de café, suplementados com glicose. Os experimentos foram realizados em frascos Erlenmeyer de 250 mL. As condicões experimentais foram: umidade inicial de 70%, adicão de 20% de glicose e pH 6,0. Os frascos foram cobertos com gaze e a aeracão ocorreu por difusão passiva. A análise do headspace da cultura foi feita por cromatografia gasosa e 12 compostos foram detectados utilizando a casca de café. A análise respirométrica foi realizada para o acompanhamento do crescimento do microrganismo pela determinacão do dióxido de carbono produzido. A producão de ésteres caracterizou o aroma frutal da cultura. A concentracão máxima de voláteis totais foi alcancada após 72 h de cultivo em casca de café (28 µmol.L-1.g-1). Os principais compostos produzidos foram acetato de etila, etanol e acetaldeído, representando 84,7%, 7,6% and 2,0% dos voláteis totais, respectivamente.