Inoculum Concentration Research Articles

The Optimization, Kinetics Model, and Lab-Scale Assessments of Phenol Biodegradation Using Batch and Continuous Culture Systems

Phenol was considered a severe hazard to all ecosystems even at low concentrations. The bioremediation process is an eco-friendly process for complete phenol degradation and bioelectricity generation. In the present study, a consortium of native isolates was used for phenol biodegradation and bioenergy generation using nano-graphite electrodes. The optimization of nutritional and environmental parameters using batch culture revealed that the optimum conditions for maximum phenol degradation and energy generation were inoculum concentration, 1%; incubation period, 48 h; phenol, 6 ppm; MgSO4, 70 mg/L; K2HPO4, 175 mg/L; and CaCl2, 1 mg/L. Phenol biodegradation reached 93.34% with a power density of 109.419 mW/cm3. A lab-scale bioreactor was used as a continuous culture with aeration rate, agitation speed, and dissolved oxygen of 0.5 v/v/m, 750 rpm, and 30%, respectively. On using the continuous culture, phenol biodegradation and bioenergy production reached 97.8% and 0.382 W/cm3, respectively. A kinetics study using Haldane’s kinetics model reported the best fit to achieve a significant correlation coefficient (R2) value (0.9865) reaching maximum specific growth rate with initial phenol concentration of approximately 9 mg L−1 where the specific growth rates (μ, h−1) varied with different initial phenol concentrations. In conclusion, the native isolated consortium could be considered as an economical and sustainable approach to phenol biodegradation in industrial wastewater as well as bioelectricity generation.

Biochemical Characterization of Microbial Isolates and Effect of Concentration and Incubation Time of Plant and Cow Milk Yoghurt using Response Surface Methodology

The objective of the research was to evaluate the biochemical characteristics of the various microbial isolates used in the production of plant based yoghurt, the optimal growth temperatures and the effect of incubation time and inoculum concentration on the taste and desirability of the yoghurt. Pure cultures of five (5) microbial strains (Lactobacillus acidophilus; Lactobacillus bulgaricus; Lactobacillus casei; Lactobacillus lactis; Streptococcus thermophilus) were obtained from commercial yoghurt and evaluated for their biochemical characteristics, bacterial growth temperatures and synersis. The isolated organisms were used to ferment plant-based milk product produced from blends of tigernut, Dates and Coconut. Response Surface Methodology (Face centered composite design) was used to obtain an optimized ratio formulation of 0.167 for coconut, 0.667 fortigernut and 0.167 for dates. Bacterial growth temperatures were evaluated at 4°C, 27°C, 37°C,40°C, 45°C and 60°C respectively. Results showed that full bacterial growth was noticed at temperatures of 40-45°C. The taste of the yoghurt improved from 6.58 to 6.71 when the incubation temperature changed from about 45°C to 44°C and the duration of incubation changed from 15.4 hrs to 14.7 hrs.

Biotransformation of Deoxynivalenol by a Dual-Member Bacterial Consortium Isolated from Tenebrio molitor Larval Feces.

In this study, a dual-member bacterial consortium with the ability to oxidize deoxynivalenol (DON) to 3-keto-DON, designated SD, was first screened from the feces of Tenebrio molitor larvae. This consortium consisted of Pseudomonas sp. SD17-1 and Devosia sp. SD17-2, as determined by 16S rRNA-based phylogenetic analysis. A temperature of 30 °C, a pH of 8.0-9.0, and an initial inoculum concentration ratio of Devosia to Pseudomonas of 0.1 were optimal single-factor parameters for the DON oxidation activity of the bacterial consortium SD. Genome-based bioinformatics analysis revealed the presence of an intact PQQ biosynthesis operon (pqqFABCDEG) and four putative pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) genes in the genomes of Pseudomonas strain SD17-1 and Devosia strain SD17-2, respectively. Biochemical analyses further confirmed the PQQ-producing phenotype of Pseudomonas and the DON-oxidizing enzymatic activities of two of four PQQ-dependent ADHs in Devosia. The addition of PQQ-containing a cell-free fermentation supernatant from Pseudomonas activated DON-oxidizing activity of Devosia. In summary, as members of the bacterial consortium SD, Pseudomonas and Devosia play indispensable and complementary roles in SD's oxidation of DON. Specifically, Pseudomonas is responsible for producing the necessary PQQ cofactor, whereas Devosia expresses the PQQ-dependent DON dehydrogenase, together facilitating the oxidation of DON.

Variability of Sarocladium oryzae [(Sawada) Games & Hawksworth] and identification of novel donors for sheath rot resistance among temperate germplasm lines of rice

AbstractSheath rot is one of the most destructive emerging diseases of rice in Asia and a significant loss (20–85%) to rice production is caused by this disease under temperate agro-climatic conditions of Kashmir. Disease is more prevalent in higher belts of valley where Japonica rice cultivation is more common. Also, late-maturing varieties are more likely to be attacked by sheath rot disease of rice. Information regarding the occurrence and distribution of any pathogen and its variability in the field is very much essential to devise a suitable disease management programme against the particular pathogen. The isolates of Sarocladium oryzae tested in the present study exhibited considerable variation in cultural characteristics, morphology and pathogenicity. The resistant sources for sheath rot disease of rice were identified among the temperate germplasm of rice under a relatively high inoculum concentration and disease pressure of all the variable isolates of S. oryzae. Among 219 rice genotypes screened for resistance against S. oryzae under field conditions, 42 genotypes were further evaluated under controlled epiphytotic conditions against all the 18 isolates of S. oryzae. Four genotypes (SKUA-354, ORN-V4, GS-183, GS357) were finally selected with high resistance against sheath rot disease of rice which can be utilized as potential donors for the development of disease-resistant varieties of rice against sheath rot.

Isolation and Characterization of the Wastewater Micropollutant Phenacetin-Degrading Bacterium Rhodococcus sp. Strain PNT-23.

Phenacetin, an antipyretic and analgesic drug, poses a serious health risk to both humans and aquatic organisms, which is of concern since this micropollutant is frequently detected in various aquatic environments. However, rare pure bacterial cultures have been reported to degrade phenacetin. Therefore, in this study, the novel phenacetin-degrading strain PNT-23 was isolated from municipal wastewater and identified as a Rhodococcus sp. based on its morphology and 16S rRNA gene sequencing. The isolated strain could completely degrade 100 mg/L phenacetin at an inoculum concentration of OD600 1.5 within 80 h, utilizing the micropollutant as its sole carbon source for growth. Strain PNT-23 exhibited optimal growth in LB medium at 37 °C and a pH of 7.0 with 1% NaCl, while the optimal degradation conditions in minimal medium were 30 °C and a pH of 7.0 with 1% NaCl. Two key intermediates were identified during phenacetin biodegradation by the strain PNT-23: N-acetyl-4-aminophenol and 4-aminophenol. This study provides novel insights into the biodegradation of phenacetin using a pure bacterium culture, expands the known substrate spectra of Rhodococcus strains and presents a potential new candidate for the microbial removal of phenacetin in a diverse range of environments.

Selection of lactic acid bacteria as biopreservation agents and optimization of their mode of application for the control of Listeria monocytogenes in ready-to-eat cooked meat products

In order to meet consumers´ demands for more natural foods and to find new methods to control foodborne pathogens in them, research is currently being focused on alternative preservation approaches, such as biopreservation with lactic acid bacteria (LAB). Here, a collection of lactic acid bacteria (LAB) isolates was characterized to identify potential biopreservative agents. Six isolates (one Lactococcus lactis, one Lacticaseibacillus paracasei and four Lactiplantibacillus plantarum) were selected based on their antimicrobial activity in in vitro assays. Whole genome sequencing showed that none of the six LAB isolates carried known virulence factors or acquired antimicrobial resistance genes, and that the L. lactis isolate was potentially a nisin Z producer. Growth of L. monocytogenes was successfully limited by L. lactis ULE383, L. paracasei ULE721 and L. plantarum ULE1599 throughout the shelf-life of cooked ham, meatloaf and roasted pork shoulder. These LAB isolates were also applied individually or as a cocktail at different inoculum concentrations (4, 6 and 8 log10 CFU/g) in challenge test studies involving cooked ham, showing a stronger anti-Listerial activity when a cocktail was used at 8 log10 CFU/g. Thus, a reduction of up to ~5.0 log10 CFU/g in L. monocytogenes growth potential was attained in cooked ham packaged under vacuum, modified atmosphere packaging or vacuum followed by high pressure processing (HPP). Only minor changes in color and texture were induced, although there was a significant acidification of the product when the LAB cultures were applied. Remarkably, this acidification was delayed when HPP was applied to the LAB inoculated batches. Metataxonomic analyses showed that the LAB cocktail was able to grow in the cooked ham and outcompete the indigenous microbiota, including spoilage microorganisms such as Brochothrix. Moreover, none of the batches were considered unacceptable in a sensory evaluation.Overall, this study shows the favourable antilisterial activity of the cocktail of LAB employed, with the combination of HPP and LAB achieving a complete inhibition of the pathogen with no detrimental effects in physico-chemical or sensorial evaluations, highlighting the usefulness of biopreservation approaches involving LAB for enhancing the safety of cooked meat products.

Study of prebiotic effect of pineapple crown powder on development of white finger millet vegan probiotic beverage

The present study aimed to develop a new probiotic functional beverage using white finger millet (WFM), pineapple crown powder (PCP), and Lactobacillus rhamnosus GG NCDC 347 (LGG) as probiotic microbe. The beverage was optimized using CCD taking PCP concentration (1–3%) and inoculum concentration (1–3%) as independent variables and pH, viable probiotic, and viscosity as dependent variables. At 2% PCP with 1% (v/v) LGG, obtained the optimized white finger millet probiotic beverage (OWMPB). The OWMPB showed an overall increase in nutritional composition, total phenolics (10.02 mg GAE/g), flavonoid content (8.24 mg QE/g), antioxidant, physiochemical and functional properties compared to the control beverage (without PCP). To determine the effect of the addition of PCP to the beverage, GC/MS, E-nose, FTIR, and HPLC analysis were done, which showed the presence of new phytochemical, functional, and volatile compounds, having therapeutic benefits. Thus, PCP and WFM can be used efficiently in developing a probiotic beverage.

Using Central Composite Design to Improve Methane Production from Anaerobic Digestion of Tomato Plant Waste

Tomato plant waste (TPW) is a significant agricultural byproduct that has been often underutilized. Recent studies have shown that its use to obtain methane in an anaerobic digestion (AD) process is viable. However, there is not much information available on studies to improve methane production from this substrate using statistical methods for optimization processes such as central composite design (CCD). For this investigation, CCD was adopted to analyze the effect of S/I ratio (substrate/inoculum ratio) (0.32–1.12), temperature (27–43 °C), and inoculum concentration (10.35–20.95 g VS/L) on methane generation and volatile solids (VS) removal in a batch AD system mono-digestion of TPW. The highest average value of methane yield was found to be 210.8 mL CH4/g VS (S/I ratio 0.48, 40 °C, and 18.80 g VS/L), and the highest average value of VS removal was found to be 36.9% (S/I ratio 1.12, 35 °C, and 15.65 g VS/L). We obtain a model with a better fit for the VS removal (R2 = 0.9587) than for the methane production (R2 = 0.9156). Temperature and S/I ratio were the factors most important for methane production and VS removal, respectively.

Optimization of Factors for the Biological Treatment of Free and Complexed Cyanide

Mexico is characterized as a mining country since it is the world’s main silver producer. During its extraction, wastewater (mining tailings) is generated which contains cyanide and heavy metals. The purpose of this research was to determine whether a bacterial consortium isolated from a tailings dam can use cyanide as a source of nitrogen and carbon to carry out its biodegradation. The study determined the effects of three physicochemical factors (pH, temperature and inoculum concentration) and three metals (copper, iron and nickel) on cyanide biodegradation. The results showed that the highest cyanide removals were obtained when working with a pH of 9.5, a temperature of 25 °C and 15% v/v of inoculum (88%), while the optimum values for copper, iron and nickel were 0, 7.7 and 0.46 mg·L−1, respectively, showing that copper causes an inhibitory effect (cyanide biodegradation of 68%) on the bacteria and consequently on the biological degradation of cyanide and that iron can promote the biodegradation of the pollutant by 91%.

Drosophila melanogaster as a model of systemic dermatophytosis.

Dermatophytosis is one of the most common fungal infections worldwide. The distribution of dermatophytes varies across continents, but the genera Trichophyton and Microsporum have emerged as the main isolated agents in humans and animals. To validate Drosophila melanogaster flies as a fast and feasible model to study dermatophytic infections. Wild-type (WT) and Toll-deficient D. melanogaster flies were infected by Trichophyton rubrum, T. mentagrophytes, Microsporum canis and Nannizzia gypsea by pricking with a needle previously dipped in inoculum concentrations ranging from 103 to 108 colony-forming units/mL. Establishment of infection was confirmed by survival curves, histopathological analysis and fungal burden. Thereafter, flies were treated with terbinafine, itraconazole and clioquinol. WT flies were predominantly resistant to the infection, whereas Toll-deficient flies succumbed to the four dermatophyte genera tested. The antifungal drugs protected flies from the infection, except for N. gypsea whose survival curves did not differ from the untreated group. This pilot study confirms that D. melanogaster is a suitable model to study the virulence and antifungal drug efficacy in dermatophyte species.

The responding mechanism of indigenous bacteria in municipal wastewater inoculated with different concentrations of exogenous microalgae

Indigenous bacteria popularly exist in real wastewater. Therefore, the potential interaction between bacteria and microalgae is inevitable in microalgae-based wastewater treatment systems. It is likely to affect the performance of systems. Accordingly, the characteristics of indigenous bacteria is worth serious concerning. Here we investigated the response of indigenous bacterial communities to variant inoculum concentrations of Chlorococcum sp. GD in municipal wastewater treatment systems. The removal efficiency of COD, ammonium and total phosphorus were 92.50%–95.55%, 98.00%–98.69%, and 67.80%–84.72%, respectively. The bacterial community responded differently to different microalgal inoculum concentrations, which was mainly affected by microalgal number, ammonium and nitrate. Besides, there were differential co-occurrence patterns and carbon and nitrogen metabolic function of indigenous bacterial communities. All these results indicated that bacterial communities responded significantly to environmental changes caused by the change of microalgal inoculum concentrations. The response of bacterial communities to different microalgal inoculum concentrations was beneficial for forming a stable symbiotic community of both microalgae and bacteria to remove pollutants in wastewater.

Optimization of biogas production from anaerobic co-digestion of fish waste and water hyacinth

Many fresh water bodies face a great challenge of an invasive weed called water hyacinth (WH) which has great impacts on the environment, ecology, and society. Food and Agriculture Organization (FAO) estimates that over nine million tons of Fish wastes (FW) are thrown away each year. The fish waste generated poses environmental and health hazards because in most cases it is either disposed into pits or discarded onto the open grounds. Both WH and FW are potential substrates for biogas production. However, utilization of FW substrate alone has a limitation of producing a lot of amounts of volatile fatty acids (VFAs) and ammonia. Their accumulation in the digester inhibits substrate digestion. Consequently, as stand-alone it is not suitable for anaerobic digestion (AD). This can be overcome by co-digestion with a substrate like WH which has high carbon to nitrogen (C/N) ratio prior to biodigestion. Experimental variable levels for biogas were substrate ratio (WH:FW, 25–75 g), inoculum concentration (IC, 5–15 g/250 mL), and dilution (85–95 mL). Design-Expert 13 was used for optimization and results analysis. Response surface methodology (RSM) was used to examine the effects of operating parameters and identify optimum values for biogas yield. Optimum values for maximum biogas with the highest methane yield of 68% were found to be WH:FW ratio, 25:75 g, 15 g of IC, and 95 mL for dilution. The yield was 16% and 32% greater than FW and WH mono-digestion, respectively. The biogas yield was expressed as a function of operating variables using a quadratic equation. The model was significant (P < 0.05). All factors had significant linear and quadratic effects on biogas while only the interaction effects of the two factors were significant. The coefficient of determination (R2) of 99.9% confirmed the good fit of the model with experimental variables.

Kinetics of biomass and polyhydroxyalkanoates synthesis using sugar industry waste as carbon substrate by Alcaligenes sp. NCIM 5085

Aim: o optimize the operating parameters and further determine the kinetics of biomass and polyhydroxyalkanoate synthesis using sugar industry waste as a carbon substrate by Alcaligenes sp. NCIM 5085. Methodology: Molasses, clarified and pretreated with sulphuric acid, was taken in a batch reactor for fermentation to generate biomass and further for polyhydroxyalkanoate synthesis where Alcaligenes sp. NCIM 5085 was used as a culture. A number of parameters were observed during fermentation experiments to optimize the biomass and PHA namely: initial substrate concentration, incubation period, C/N ratio, inoculum concentration and pH. Fermented solution was filtered to account the yield of biomass from the solution at each time interval decided for each experiment and polyhydroxyalkanoate was extracted from the biomass by chloroform and methanol extraction. Growth kinetics of biomass was modeled by Monod and Logistic models whereas Luedeking-Piret model was used for polyhydroxyalkanoate production rates. Results: From the fermentation experiments, it was found that for 40 g l-1 of molasses at neutral pH, 4.48 g l-1 of biomass was synthesized when fermentation was observed for 48 hr. One percent of cell dry weight of biomass was extracted as polyhydroxyalkanoate. Logistic model for growth kinetics and Luedeking-Piret model for polyhydroxyalkanoate production rate were found in agreement with the experiments where regression coefficient was maximum with minimum error. Interpretation: The PHA growth kinetics and PHA production kinetics done in this work can be utilized to design a bioreactor for PHA production at industrial scale. Key words: Alcaligenes sp., Biomass, Cane molasses, Growth kinetics, Polyhydroxyalkanoates

Parafilm as an efficient transport matrix for corneal scrapings.

Acanthamoeba spp. are free-living parasites increasingly implicated in causing Acanthamoeba keratitis (AK). AK is diagnosed by demonstration of parasites in corneal samples by direct microscopy, culture, and nucleic acid amplification. Most commonly, corneal scrapings are sent to the laboratory smeared between two glass slides. These scrapings are suitable for direct microscopy but less suitable for culture and polymerase chain reaction (PCR) which, in turn, are more sensitive for the diagnosis of AK. The aim of the study was to explore better alternatives for transporting corneal scrapings from the point-of-care eye center to the concerned laboratories. The study used small Parafilm (Bemis Company Inc., USA) squares (PSs) of 1 cm each prepared by cutting Parafilm using a surgical blade under sterile conditions. Each of the four different dilutions of Acanthamoeba suspension (15, 30, 60, and 120 cells) was used in this study. Each dilution was added onto the surface of 36 PSs and kept at room temperature for 24-h, 48-h, and 72-h incubation. The PSs for one particular time point and dilution were used for calcofluor white staining, its inoculation onto the surface of nonnutrient agar having a lawn of Escherichia coli, and Acanthamoeba-specific PCR amplification. In addition, two PSs inoculated with 30 cells and incubated for 24 h and 72 h were used for scanning electron microscopy (SEM). All three diagnostic techniques, i.e. microscopy, culture, and PCR, detected the presence of Acanthamoeba at all the tested concentrations and time points. However, the growth pattern on culture changed directly in proportion to increased incubation periods and increased concentration of inoculum. In addition, the adherence of Acanthamoeba to the Parafilm was confirmed by SEM; these results suggest the use of these PSs as a suitable matrix for the transport of corneal scrapings.

Plant growth stage and Phoma medicaginis inoculum concentration together determine severity of Phoma black stem and leaf spot and consequent phytoestrogen production in annual Medicago spp.

AbstractPhoma black stem and leaf spot disease (Phoma medicaginis) not only destroys annual Medicago spp. forage and seed yield but also reduces herbage quality by consequent phytoestrogen production that reduces ovulation of grazing animals. Two controlled environment studies evaluated the effects of plant developmental stage in annual Medicago rugosa ‘Paraponto’ and M. scutellata ‘Sava’ and different inoculum concentrations of P. medicaginis in M. littoralis ‘Harbinger’ and M. polymorpha ‘Serena’ on disease development and coumestrol production. Disease incidence and severity and coumestrol production were dependent on plant developmental stage, cultivar and inoculum level (all p ≤ 0.001). Disease was least in 4‐week‐old plants; highest coumestrol was in inoculated 10‐week‐old Sava (1353 mg/kg) and least coumestrol in uninoculated 4‐week‐old Paraponto (87 mg/kg); and there was a positive correlation of disease incidence/severity factors with coumestrol across cultivars and plant growth stages (p &lt; 0.001). Disease levels and coumestrol production were determined by inoculum concentration and cultivar (both p ≤ 0.001). Highest coumestrol was in Serena inoculated with 107 conidia/mL (265 mg/kg); lowest coumestrol was in uninoculated Harbinger (6 mg/kg); and there was a significant positive correlation of disease incidence/severity factors with coumestrol across cultivars and inoculum concentrations (p &lt; 0.001). These studies emphasize both the opportunity for farmers to better use annual Medicago spp. stands for grazing reproducing animals early in the growing season when both disease and coumestrol levels are lowest, and the need for heightened farmer vigilance at later growth stages with greater disease and consequent phytoestrogen risk for grazing animals.

Optimization of Fermentation for Bioethanol Production from sour Citrus aurantifolia Fruit Juice using Natural Palmyrah Toddy Yeast

&#x0D; Large scale consumption of fossil fuel to meet the increasing demand causes adverse effects on the environment due to the emission of harmful greenhouse gases. The production of bioethanol from diverse fruit juices that are underutilized because of poor taste quality could be one of the alternative fuels to overcome the issues. The objective of the study was to produce bioethanol from sour Citrus aurantifolia fruit juice using palmyrah toddy yeast and to optimize the conditions to increase the bioethanol yield. The sour Citrus fruit juice was inoculated with palmyrah toddy yeast (Saccharomyces cerevisiae - 2g/L) in the fermentation media (100ml, sour Citrus fruit juice: distilled water = 1:3) composed of 10 g/L yeast extract, 10 g/L KH2PO4, 2 g/L (NH4)2SO4, 2 g/L peptone and 0.5 g/L MgSO4·7H2O and allowed for fermentation for 24 hours at room temperature. The amount of ethanol produced from the Citrus juice was 0.8% (V/V) after 24 hours of fermentation. In order to optimize the fermentation process for Citrus aurantifolia, a variety of experimental parameters were studied, including the type of nitrogen source (ammonium sulphate, ammonium nitrate, ammonium chloride, and urea), inoculum size (0.4 to 1.0 g/100 mL), temperature (20 to 40 °C), rotation speed (50 to 250 rpm), concentration of raw fruit juice (5 to 100%), amount of urea (0.1 to 2.0 g/100 mL), carbon source (glucose, sucrose, maltose, and dextrose), amount of sucrose (1 to 20 g), pH of the medium (3.0 to 8.0), and incubation period (24 to 96 h). After conducting the experiments, it was found that using Citrus aurantifolia at 100% concentration resulted in significantly higher ethanol yields of 11.50%, which was 14.37 times higher than the non-optimized conditions. The highest yield was achieved when the fermentation was carried out at 35 °C for 24 h with an inoculum concentration of 0.5 g/100 mL, a rotation speed of 150 rpm, a pH of 4.0, 0.1 g/100 mL urea as the nitrogen source, and 10 g/100 mL sucrose as the carbon source. Large scale fermentation study using bioreactor should be done to determine whether this finding could be commercialized.&#x0D; Key words: Bioethanol, Fermentation, Palmyrah toddy yeast, Citrus fruit juice, Urea

Alternative Development and Processing of Fermented Beverage and Tempeh Using Green Beans from Four Genotypes of Lupinus mutabilis

In this study, the nutritional characteristics of two formulations were studied: A fermented beverage and tempeh. Green seeds from four different cultivars of tarwi (Lupinus mutabilis) were used: Andino, Guaranguito, Ecuadorian, and Peruvian. The seeds were first debittered, and then, they were fermented, using two inoculum doses and two fermentation times. For the fermented beverage, Saccaromyces cerevisae was used at doses of 1.5 and 2.5% dry weight and fermentation times of 72 and 96 h. For the tempeh, the inoculum used was the Rhizopus oligosporus strain NRRL 2710 at doses of 0.015 and 0.025% dry weight and fermentation times of 48 and 72 h. The alcoholic strength, total solid soluble, and pH in the fermented beverage were analyzed, while proximal analysis was performed for both the fermented beverage and tempeh. The results indicate that, in all cases, the Andino cultivar produced a beverage with higher alcoholic strength, regardless of the inoculum concentration and fermentation time. In both the fermented beverage and tempeh, a varietal effect was found for protein and crude fiber. The Andino cultivar had the highest protein, fat, and fiber content. The alkaloid content was similar in the beverage and tempeh, but the effect of fermentation was markedly increased with the higher inoculum dose and longer fermentation time. The alkaloid content was highest in the Peruvian cultivar, while it was similar in the remaining three cultivars. Tarwi grains have a high nutritional value, but the grain is traditionally used dry. In this study, we used green grain, which contains lower alkaloid content. Moreover, the crop cycle is shortened, and it can be used in the manufacture of fermented drinks and tempeh. In this way, we show that it is valid to use green grain instead of dried grain, constituting an improvement in the traditional use of lupine grain, and our study shows that it is possible to make both fermented drink and tempeh with green grain, showing the influence of the genotype and dosage starter.

Optimization of Ustilago nuda Inoculum Concentration for Screening Un8-Mediated Loose Smut Resistance in Barley Reveals a Resistance Reaction that Disrupts Seed Germination and Suggests a Role for Abscisic Acid in Disease Development.

Barley loose smut has been effectively controlled for decades through resistance conferred by the Un8 gene. However, evaluation of loose smut reaction using floret inoculation at the standard inoculum concentration is associated with the production of small, discolored seeds in Un8 carriers and susceptible genotypes. Interestingly, Un8 carriers also displayed significantly poorer germination than susceptible genotypes and produce short-lived seedlings following inoculation. To understand these observations, a Un8 carrier (TR11698) and susceptible non-Un8 carrier (CDC Austenson) were assessed for seed traits, Ustilago nuda biomass in the seed, infection rate, and phytohormone profile across a range of lower inoculum concentrations. At lower inoculum concentrations, seed appearance and weight improved in both genotypes, and infection rate increased in CDC Austenson. Pathogen load in the seed was similar in both genotypes and was positively correlated with the CDC Austenson infection rate. No infection was ever observed in TR11698. Significantly, germination rate improved in CDC Austenson, whereas the very low germination rate and short-lived seedlings remained associated with TR11698. It appears that poor seed appearance in both genotypes and low germination rate in the susceptible genotype can be improved by lowering the inoculum concentration. However, the very low germination rates and seedling death associated with the Un8 carrier TR11698 are indicative of Un8-mediated resistance to loose smut. Finally, profiling of 38 phytohormones revealedthat larger seeds observed at some inoculum concentrations compared with mock inoculation had higher abscisic acid concentrations. This could represent a pathogen survival strategy by ensuring better growth of the host.

Publisher’s Note

A report from the FAO (Food and Agriculture Organization) estimates that 9.1 million tons of Fish wastes (FW) are thrown away each year. Water hyacinth (WH) is the most invasive water weed that thrives in freshwater water and has impacts on the environment, ecology, and society. The waste generated is either discarded onto the open ground or disposed into pits which result in environmental pollution and health hazards. FW and WH can be used for biogas production. However, FW digests quickly and produces a lot of ammonia and Volatile fatty acids (VFAs) which inhibit the digestion when digested alone, it has also a low carbon to nitrogen (C/N) ratio; consequently, it is not directly suitable for anaerobic digestion (AD). In this regard, co-digestion with WH (high C/N ratio) prior to AD is necessary. To examine the effects of the operating factors: Substrate ratio (WH:FW), Inoculum concentration (IC) and Dilution, Central composite design (CCD) in Design Expert 13 software was used to design experiments of three factors at five levels which resulted in 17 runs. Laboratory scale experiments were carried out under mesophilic temperature (37 °C). The maximum biogas with the highest methane content of 68.15% was produced at the substrate ratio of 25:75 g/ 250 mL (WH:FW), 15 g/ 250 mL of IC and dilution of 95 mL. The biogas yield increased by 16.1% and 32.4% compared to pure WH and FW respectively. This implied that the operating conditions were efficient in maximising the biogas yield. FW had a lower C/N ratio; further study needs to consider co-digestion with other higher C/N ratio substrates.

Comparative Study of Inoculation Methods to Determine the Aggressiveness of Xanthomonas citri pv. glycines Isolates and to Evaluate the Reaction of Soybean Cultivars to Bacterial Pustule

Bacterial pustule caused by Xanthomonas citri pv. glycines (syn. X. axonopodis pv. glycines) is an important bacterial disease for soybean production worldwide. Currently, the planting of resistant soybean cultivars is the main measure for control of the disease. Therefore, the objectives of this study were to determine the aggressiveness of different X. citri pv. glycines isolates and to establish a new protocol to evaluate the reaction of soybean cultivars to bacterial pustule under greenhouse conditions. The molecular analysis based on the entire 16S rRNA gene sequence revealed that the isolates GDM 01, GDM 02, 333 and 87-2 belong to the X. citri pv. glycines pathovar with 100% identity with the pathotype strain K29 of this bacterium. Differences in aggressiveness were observed among the X. citri pv. glycines isolates. There were differences in the reaction of the soybean cultivars to bacterial pustule, depending on the inoculation method. The isolates 333, GDM 01 and GDM 02 were the most aggressive, while 87-2 and GDM 03 were the least aggressive ones. The best conditions for inoculation of the soybean plants were in the V3 vegetative growth stage with the inoculum concentration of 108 CFU mL−1, and moist chamber for 24 h before and after inoculation. Spraying without injury was the quickest and most practical inoculation method to screen soybean genotypes for resistance to bacterial pustule in breeding programs. Further, this method of inoculation closely simulates the natural bacterial infection under field conditions and produces typical symptoms of the disease. The standard brush inoculation method overestimated disease severity, so we do not recommend this method for the evaluation of bacterial pustule resistance in soybean breeding programs.