Type Of Inoculum Research Articles

Changing the Inoculum Type From Preculture to Spore Suspension Markedly Alters the Production of Secondary Metabolites in Filamentous Microbial Coculture

The shake flask cocultures of Aspergillus terreus and Streptomyces rimosus were investigated with regard to the production of mevinolinic acid (lovastatin), oxytetracycline, and other secondary metabolites (SMs). The aim of the study was to determine the effect of inoculum type (spore suspension or preculture) on the levels of SMs in the fermentation broth. Altogether, 17 SMs were detected, including 4 products with confirmed identities, 10 putatively annotated metabolites, and 3 unknown molecules. As observed over the course of qualitative and quantitative analyses, the selection of inoculum type markedly influenced the SM-related outcomes of cocultures. Depending on the coculture initiation procedure, replacing the preculture with spore inoculum positively affected the biosynthesis of oxytetracycline, butyrolactone I, (+)-geodin, as well as the molecules putatively identified as rimocidin, CE-108, and (+)-erdin. It was concluded that the comparative analyses of SM production in filamentous microbial cocultures and monocultures are dependent on the type of inoculum and thus the diversification of inocula is highly recommended in such studies. Furthermore, it was demonstrated that designing a coculture experiment that involves only a single type of inoculum may lead to the underestimation of biosynthetic repertoires of filamentous microorganisms.

Anthracocystis panici-leucophaei: A Potential Biological Control Agent for the Grassy Weed Digitaria insularis

Anthracocystis panici-leucophaei, causal agent of smut on Digitaria insularis (sourgrass), was evaluated as a biological control agent for this weed. Two types of inocula (teliospore and sporidia) were tested to assess its infectivity. The effects of teliospore and sporidia inoculations at different phenological stages of sourgrass were compared, as well as the potential of sporidia and teliospores in post-emergence sourgrass management. Virulence tests were conducted with the isolates obtained from D. insularis and evaluation of specificity of A. panici-leucophaei. Both teliospores and sporidia of A. panici-leucophaei are infective to D. insularis in three different phenological stages. Newly emerged plants with one pair of leaves are more sensitive to A. panici-leucophaei. Infection by A. panici-leucophaei inhibits the growth of sourgrass, decreasing several physiological parameters of D. insularis plants. The fungus produces systematic infection of sourgrass plants and may induce the formation of sori in a significant proportion of the plant panicles, partly castrating those plants. Among sixteen A. panici-peucophaei isolates tested, isolate 46 was the most virulent and inhibited the growth of sourgrass plants, and thus appears to have good potential as a biological control agent to be deployed against sourgrass. A. panici-leucophaei was demonstrated to be specific to D. insularis.

Differences between Pseudobombax grandiflorum and Bauhinia forficata in terms of responsiveness and dependence to mycorrhiza

ABSTRACT The benefits promoted by arbuscular mycorrhizal fungi (AMF) to forest species seedlings include higher growth rate, better nutrition, and higher survival rates. Inoculation with AMF may facilitate revegetation of degraded lands, although it depends on symbionts and environmental conditions, such as soil P availability. In this sense, the lack of information justifies the carrying out of studies of this nature. We investigated the dependence and responsiveness of two forest species native to the Atlantic Forest, Pseudobombax grandiflorum and Bauhinia forficata, to different AMF inocula (isolated AMF species, Dentiscutata heterogama, DH, Gigaspora margarita, GM, Rhizophagus clarus, RC, mixed inoculum with these former three AMF species, MI) compared to the uninoculated control (UC), combined with different P doses applied to the substrate (0, 24, 71, 213, and 650 mg kg -1 ), under greenhouse conditions. We evaluated root colonization, growth, and nutritional variables for Pseudobombax grandiflorum and Bauhinia forficata, 112 and 116 days after sowing with pre-germinated seeds, respectively. Native forest species exhibited different degrees of mycorrhizal dependence. The highest mycorrhizal dependence of P. grandiflorum seedlings was indicated by significant benefits, both growth and nutritional, promoted by innoculation treatments, under fertilization with the intermediate dose of P (213 mg kg -1 ). In fact, under this P dose, seedlings responded to a maximum increase in biomass in the GM treatment and maximum concentration of P and N in the shoots in the UC and DH treatments, respectively. The lowest mycorrhizal dependence of B. forficata seedlings was highlighted by significant growth benefits promoted by inoculation treatments under the lowest doses of P (24 or 71 mg kg -1 ). Under this P dose, we observed seedlings with maximum value of the root:shoot ratio in most of the types of inoculation and also higher values of biomass and height in the MI treatment. The responsiveness in terms of increase in growth and nutritional variables varied depending on the forest species, the dose of P applied to the substrate, and the AMF type of inoculum used. Root biomass and total dry biomass, mainly, should preferably be included in future studies with the same objective as the present study, as they were more relevant to point out the differences between treatments, in comparison with the variables associated with nutritional variables.

Regional Variability of Chestnut (Castanea sativa) Tolerance Toward Blight Disease.

Since its introduction into Europe in the first half of the 20th century, Cryphonectria parasitica has been gradually spreading across the natural range of the sweet chestnut (Castanea sativa Mill.), infecting the trees and causing lethal bark cankers. Serendipitously, a hyperparasitic Cryphonectria hypovirus 1 (CHV1), which attenuates C. parasitica virulence in combination with more tolerant European chestnut species, was able to ward off the worst effect of the disease. In North America, unfortunately, the native Castanea dentata is now functionally extinct since it occurs only as root sprouts in eastern deciduous forests where it was once dominant. In our work, we investigated changes in C. parasitica populations over time and the regional variability in chestnut populations' tolerance toward the blight disease. While vegetative compatibility (vc) type diversity and prevalence of hypovirulence remained similar as in previous studies, in the Buje population, unlike in previous studies, we were unable to find any hypovirulent fungal strains. The most common vegetative compatibility types (vc types) were EU-1, EU-2 and EU-12. However, several rare EU-types were found, including one previously unreported: EU-46. By inoculating several C. parasitica strains on tree stems from several chestnut populations, we observed that the induced lesion size was affected by the type of inoculum (CHV1-free or CHV1-infected), genotype-related individual chestnut stem and chestnut stem population of origin-related variability. The largest lesions were induced by CHV1-free fungal isolate DOB-G: 20.13 cm2 (95% C.I. 18.10-22.15) and the smallest by CHV1-infected L14/EP713: 2.49 cm2 (95% C.I. 1.59-3.39). Surprisingly, the size of the lesions induced by other CHV1-infected strains fell somewhere in between these extremes. The size of induced lesions was dependent on the population of origin as well and ranged from 11.60 cm2 (95% C.I. 9.87-13.33) for stems from the Moslavačka gora population to 17.75 cm2 (95% C.I. 15.63-19.87) for stems from Ozalj.

Predicting biohydrogen production from dark fermentation of organic waste biomass using multilayer perceptron artificial neural network (MLP–ANN)

The focus on sustainable energy has increased interest in biohydrogen production through dark fermentation of organic waste biomass, offering dual benefits of energy production and waste management. Optimizing this process is challenging due to complex interactions among substrate composition, microbial consortia, and fermentation parameters. A multilayer perceptron artificial neural network model was developed to predict biohydrogen yield from organic waste. The model, trained on 180 data points from 35 studies, uses inputs, such as substrate type, inoculum type, concentration, pH, and temperature, with hydrogen yield as the output. The multilayer perceptron artificial neural network model achieved high accuracy, with a root mean square error of 0.3838, a mean absolute percentage error of 0.1938, and a coefficient of determination of 0.8381. These results demonstrate the model's effectiveness in predicting biohydrogen production, providing a valuable tool for optimizing the fermentation process and advancing sustainable energy solutions.

Enhancing the biodegradation of hydrophobic volatile organic compounds: A study on microbial consortia adaptation and the role of surfactants

The emission of hydrophobic Volatile Organic Compounds (VOCs) is a serious environmental issue. Typically, biofilters (BFs) are employed for their treatment, with the potential enhancement of mass transfer through the addition of surfactants. However, disparate results in previous studies have been observed, attributed to uncontrolled conditions during the introduction of surfactants to BFs. Additionally, there has been limited exploration of microbial consortium adaptation to surfactants. To address these gaps, this study followed two approaches. First, the long-term (247 days) removal of cyclohexane was studied in a stirred tank bioreactor (STBR) inoculated with Rhodococcus erythropolys E1 and using Tween 80 at three times the critical micelle concentration (CMC). Second, the short-term (9 days) impact of two (bio)surfactants [Tween 80 (1 × CMC) and Quillaja Saponin (QS, 1 × CMC)] on the removal of cyclohexane, hexane and toluene was investigated in batch tests using three types of inocula: a pure culture of Rhodococcus erythropolys E1 (X0), a microbial consortium adapted to cyclohexane (X1), and a microbial consortium adapted to cyclohexane with Tween 80 (X2). For long-term operation, the addition of Tween 80 at 3 × CMC improved cyclohexane removal efficiency (RE) to 87 ± 1% (elimination capacity, EC = 145 ± 25 mg m−3 h−1, gas residence time, GRT = 20 min, inlet concentration, Cin = 14.9 ± 2.5 ppmv), compared to a RE of 32 ± 9% (EC = 44 ± 8 mg m−3 h−1, GRT = 20 min, Cin = 15.1 ± 0.7 ppmv) under similar conditions without surfactants. For short-term operation, the addition of QS at 1 × CMC significantly increased biomass growth, resulting in lower maximum specific consumption rates for X1 and X2 compared to scenarios without surfactants or 1 × CMC Tween 80. The most abundant genera in X1 and X2 were Paludisphaera (26-23%), 67-14 genus (17–23%), and Rhodococcus (9–18%), respectively.

Inactivation of Salmonella, Enterococcus faecium and natural microbiota on dry food matrices with microwave-driven plasma-processed air

This study explores the efficacy of plasma-processed air (PPA) as a non-thermal treatment for decontamination of dry food surfaces, such as wheat grains, black peppercorns, and onion flakes. We compared the effect of various inoculum types of Enterococcus faecium, revealing a higher resistance of the powder form compared with liquid and slurry inocula on black peppercorns. Furthermore, Enterococcus faecium NRRL B-2354 appeared to be a suitable control indicator for Salmonella. The PPA efficacy varied across matrices, being higher on medium agar (7.0 ± 0.5 log10), followed by wheat grains (2.1 ± 0.2 log10), black peppercorns (1.5 ± 0.1 log10), and no reduction on onion flakes. Variation was also found across microorganisms, as the natural microbiota was reduced on wheat grains (1.6 ± 0.6 log10 for mesophilic count and 0.8 ± 0.3 log10 for spores), while on black peppercorns this was only achieved after upscaling (1.3 ± 0.2 log10 for mesophilic and 1.5 ± 0.1 log10 for spores). The findings of this study give guidance to further optimize the application of PPA treatment by changing the technical parameter settings of the plasma setup to increase the microbial efficacy and/or combining the PPA treatment with other methods to enhance microbial control.

Microbial inoculum effects on the rumen epithelial transcriptome and rumen epimural metatranscriptome in calves

Manipulation of the rumen microbial ecosystem in early life may affect ruminal fermentation and enhance the productive performance of dairy cows. The objective of this experiment was to evaluate the effects of dosing three different types of microbial inoculum on the rumen epithelium tissue (RE) transcriptome and the rumen epimural metatranscriptome (REM) in dairy calves. For this objective, 15 Holstein bull calves were enrolled in the study at birth and assigned to three different intraruminal inoculum treatments dosed orally once weekly from three to six weeks of age. The inoculum treatments were prepared from rumen contents collected from rumen fistulated lactating cows and were either autoclaved (control; ARF), processed by differential centrifugation to create the bacterial-enriched inoculum (BE), or through gravimetric separation to create the protozoal-enriched inoculum (PE). Calves were fed 2.5 L/d pasteurized waste milk 3x/d from 0 to 7 weeks of age and texturized starter until euthanasia at 9 weeks of age, when the RE tissues were collected for transcriptome and microbial metatranscriptome analyses, from four randomly selected calves from each treatment. The different types of inoculum altered the RE transcriptome and REM. Compared to ARF, 9 genes were upregulated in the RE of BE and 92 in PE, whereas between BE and PE there were 13 genes upregulated in BE and 114 in PE. Gene ontology analysis identified enriched GO terms in biological process category between PE and ARF, with no enrichment between BE and ARF. The RE functional signature showed different KEGG pathways related to BE and ARF, and no specific KEGG pathway for PE. We observed a lower alpha diversity index for RE microbiome in ARF (observed genera and Chao1 (p < 0.05)). Five microbial genera showed a significant correlation with the changes in host gene expression: Roseburia (25 genes), Entamoeba (two genes); Anaerosinus, Lachnospira, and Succiniclasticum were each related to one gene. sPLS-DA analysis showed that RE microbial communities differ among the treatments, although the taxonomic and functional microbial profiles show different distributions. Co-expression Differential Network Analysis indicated that both BE and PE had an impact on the abundance of KEGG modules related to acyl-CoA synthesis, type VI secretion, and methanogenesis, while PE had a significant impact on KEGGs related to ectoine biosynthesis and D-xylose transport. Our study indicated that artificial dosing with different microbial inocula in early life alters not only the RE transcriptome, but also affects the REM and its functions.

EVALUATION OF BACILLUS CEREUS GROWTH IN COOKED RICE

The present study aimed to evaluate the dependence of growth dynamics of toxigenic B. cereus on rice type, inoculum type, storage time, and temperature using appropriate mathematical models. Samples of cooked white (milled) and brown (husked) rice inoculated with vegetative cell/spore suspension of B. cereus were stored for 48 h at 4 °C (correct storage) and 24 °C (inappropriate storage method). No growth of B. cereus was observed during 48 hours of storage at 4 °C and was characterised by a linear model. To assess the growth of B. cereus in the cooked rice stored at 24 °C, the Baranyi-Roberts, Gompertz and Buchanan mathematical models were used. The Gompertz model characterized the growth of B. cereus best. The type of rice and the type of inoculum did not have a significant effect on the bacterial growth. After 48 h of storage at 24°C, the B. cereus counts were in the range of 8-9 log cfu.g-1, critical value of 5 log cfu.g-1 was reached in less than 10 hours of storage. The study results indicate that cooked rice stored at 24 °C quickly becomes a health risk for consumers due to the rapid multiplication of B. cereus within a few hours. In conclusion, it is crucial to cool cooked rice immediately to 4 °C if it is not to be consumed right after cooking.

Purpureocillium lilacinum SBF054: Endophytic in Phaseolus vulgaris, Glycine max, and Helianthus annuus; Antagonistic to Rhizoctonia solani; and Virulent to Euschistus heros.

Microorganisms with multiple ecological functions can be a useful biotechnological resource in integrated pest- and disease-management programs. This work aimed to investigate the potential endophytic and virulent effects of a strain of Purpureocillium lilacinum on organic cultivation in Brazil. Specifically, the strain's ability to establish itself as an endophyte in common bean, soybean, and sunflower plants when inoculated via seed was evaluated. Furthermore, its antifungal activity against phytopathogens and its pathogenicity and virulence against insects of the order Lepidoptera, Coleoptera, and Hemiptera were evaluated. Furthermore, the strain was evaluated for its biochemical and physiological characteristics. For virulence bioassays, the experiments were conducted under a factorial scheme (2 × 3), with the following factors: (a) fungal inoculation and control without inoculum and (b) types of inocula (blastospores, aerial conidia, and metabolites). The treatments were sprayed on insect species at different stages of development. In summary, it was found that the SBF054 strain endophytically colonized the common bean, with partial recovery from the root tissues of soybean and sunflower plants, 30 days after inoculation; suppressed 86% of Rhizoctonia solani mycelial growth in an in vitro assay; and controlled eggs, nymphs, and Euschistus heros adults. These multifunctional abilities are mainly attributed to the strain's mechanisms of producing metabolites, such as organic acids, soluble nutrients, and hydrolytic enzymes.

Experimental Infection Models and Their Usefulness for White Spot Syndrome Virus (WSSV) Research in Shrimp

White spot syndrome virus (WSSV) is marked as one of the most economically devastating pathogens in shrimp aquaculture worldwide. Infection of cultured shrimp can lead to mass mortality (up to 100%). Although progress has been made, our understanding of WSSV’s infection process and the virus–host–environment interaction is far from complete. This in turn hinders the development of effective mitigation strategies against WSSV. Infection models occupy a crucial first step in the research flow that tries to elucidate the infectious disease process to develop new antiviral treatments. Moreover, since the establishment of continuous shrimp cell lines is a work in progress, the development and use of standardized in vivo infection models that reflect the host–pathogen interaction in shrimp is a necessity. This review critically examines key aspects of in vivo WSSV infection model development that are often overlooked, such as standardization, (post)larval quality, inoculum type and choice of inoculation procedure, housing conditions, and shrimp welfare considerations. Furthermore, the usefulness of experimental infection models for different lines of WSSV research will be discussed with the aim to aid researchers when choosing a suitable model for their research needs.

Salmonella and Listeria monocytogenes Survival on Field Packed Cantaloupe Contact Surfaces

Field-packing of cantaloupes involves numerous food contact surfaces that can contamination melons with foodborne pathogens; the soil on these surfaces increases throughout the harvest day. Data are lacking on the cross-contamination risk from contaminated food contact surfaces under the dry conditions typical of cantaloupe field-packing operations. This study sought to evaluate the survival of Salmonella and Listeria monocytogenes on cantaloupe field-pack food contact surfaces using both a wet and dry inoculum to provide insights into managing foodborne pathogen contamination risks. Five clean or fouled materials (cotton gloves, nitrile gloves, rubber gloves, cotton rags, and stainless steel) were inoculated with a cocktail of either Salmonella or L. monocytogenes. A wet inoculum was spot inoculated (100 µL) onto coupons. A dry inoculum was prepared by mixing wet inoculum with 100 g of sterile sand and shaking the coupons with the inoculated sand for 2 min. Coupons were held at 35°C (35% RH) and enumerated at 0, 2, 4, 6, and 8 h. Significant differences in pathogen concentrations over time were calculated, and the GInaFiT add-in tool for Excel was used to build Log-linear, Weibull, and Biphasic die-off models. Depending on the material type, coupon condition, and inoculum type, Salmonella and L. monocytogenes reductions over 8 h ranged from 0.3 to 3.3 and −0.4 to 4.2 log10 CFU/coupon, respectively. For all material types, Salmonella reductions were highest on wet-inoculated clean coupons; L. monocytogenes varied by material type. Weibull and biphasic models were a better fit of respective pathogen die-off curves than linear models. Overall, faster die-off rates were seen for wet inoculated and clean materials. Since pathogen populations remained viable over the study duration and both inoculum type and coupon condition impacted survival, frequent sanitation or replacement of food contact surfaces during the operational day is needed to reduce the risk of cross-contamination.

Design of experiments to evaluate pH and temperature parameters with different inoculums in domestic biodigester

The comprehensive management of organic urban solid waste is a concern due to its direct and indirect impact on the environment. Anaerobic Digestion (AD) has been recognized as an alternative and environmentally friendly technology for waste disposal, converting them into organic fertilizers and renewable energy. This research presents an experiment involving four reactors fed with household organic waste, three inoculated with canine, goat, and rabbit manure, and one without inoculum. The experiment was observed for 30 consecutive days to analyze the pH and temperature parameters involved in the AD process in domestic reactors. Statistical methodology, including one-way analysis of variance for assessing the effect of the type of inoculum, Tukey's simultaneous confidence intervals for mean differences, and 90 % confidence intervals for μ in temperature and manure, was utilized. Additionally, main effects analysis of the factors of average temperature and pH were conducted. The results of the one-factor experiment show that the type of inoculum does not significantly influence the variation in pH, while temperature remains relatively stable throughout the AD process. However, the analysis of main effects indicates that goat manure tends to stabilize the temperature with minimal variation, whereas variation is more heterogeneous in the other experiments.

Lactic acid production from different sources of organic solid waste: evaluation of the inoculum type and operational optimization

AbstractThis work aimed to evaluate the effect of the sources of organic solid waste on lactic acid (HLa) production. Organic fractions from cafeteria (CW), market (MW), and recycling plant (RW) wastes were used as substrates. HLa production during the self-fermentation of CW, MW, and RW, as well as during the fermentation of each substrate with anaerobic sludge (CW + sludge, MW + sludge, and RW + sludge), was evaluated in batch tests at 37 °C, initial pH of 6.5, and 150 rpm. Subsequently, the initial pH and substrate concentration of self-fermentation of CW were optimized using a central composite design. The highest HLa production was observed in the self-fermentation of CW, where several Lactobacillus species predominated in the microbial community. The higher value of HLa production using CW (20.7 g/L) was obtained at a pH of 7.29 and 115.5 gVS/L. In the optimization tests, Lactobacillus and Weissella spp. were identified as the dominant lactic acid bacteria; however, only Lactobacillus species were associated with the highest HLa production.

Acidogenic Fermentation of Food Waste for the Production of Short-Chain Fatty Acids: The Impact of Inoculum Type and Inoculum Heat Pretreatment

Acidogenic fermentation is an emerging biotechnology that allows for the utilization of food waste as a feedstock to produce high-value products such as short-chain fatty acids (SCFAs), effectively offering a tangible solution for food waste management as well as resource recovery. The objectives of the current study were to identify the ideal inoculum, waste-activated sludge (WAS) or anaerobic digester sludge (AD), for the acidogenic fermentation of food waste at room temperature, as well as to evaluate the impact of heat pretreatment of these inoculums on fermentation performance. The maximum hydrolysis yield of 399 g sCOD/kg VS added was obtained when untreated AD was used as the inoculum, whereas the pretreated AD inoculum provided the highest SCFA yield and conversion efficiency of 238 g sCODSCFA/kg VS added and 71%, respectively. Heat pretreatment had a detrimental impact on the WAS inoculum, leading to lower hydrolysis and SCFA yields, but exerted a positive influence on the AD inoculum. The microbial community showed that heat pretreatment negatively impacted the abundance of non-spore-forming hydrolytic and acidogenic microorganisms. Overall, this study demonstrates the critical role of inoculum type and heat pretreatment in optimizing the acidogenic fermentation process, laying the groundwork for future refinements in SCFA production from food waste through inoculum design.

Resistance against fungal decay of Scots pine sapwood modified with phenol-formaldehyde resins with substitution of phenol by lignin pyrolysis products

Abstract Phenol-formaldehyde (PF) resins can be impregnated and cured in situ to improve the woods dimensional stability and decay resistance. In search of renewable alternatives, the substitution of phenol by lignin cleavage products (LCP) has been discussed. However, the different chemical nature may affect the performance of the resin against fungal decay, formaldehyde emission, and equilibrium moisture content. In this study, 30 % (w/w) of the phenol in PF resins were substituted by LCP obtained from microwave-assisted pyrolysis. Scots pine sapwood was modified with the resin. The decay resistance against Rhodonia placenta, Gloeophyllum trabeum, and Trametes versicolor was determined. Additionally, effects of specimen organisation within the Petri dish, different substrates, length of leaching, and type of inoculum were studied. Further, the materials water vapor sorption properties and formaldehyde emission were determined. All modifications effectively reduced fungal decay. With 10 % weight percent gain (WPG), initial decay was detected, while 20 % WPG and 30 % WPG provided efficient protection. The substitution of phenol increases the formaldehyde emission. While further reduction in formaldehyde in the resin admixture or formaldehyde scavengers may be required, the method described herein can be used to partly replace fossil-based phenol, while maintaining good fungal resistance.

Diversity of Culturable Yeasts Associated with the Technification Level in the Process of Mezcal Production in the State of Durango

Durango State has the denomination of origin for the production of mezcal, which is made from Agave durangensis, mainly in an artisanal way; therefore, differences in the fermentation process affect the quality of the final product. The main objective of the present study was to evaluate the diversity of culturable yeasts involved in the artisanal and semi-technified process of mezcal production in the State of Durango. Three distilleries with different production processes were monitored at different fermentation stages (beginning, mid-fermentation, and end of fermentation) in the spring and summer seasons. A greater diversity was found in the distillery of Nombre de Dios in both the spring and summer production seasons (H’ = 1.464 and 1.332, respectively), since it maintains an artisanal production process. In contrast, the distillery of Durango, where a Saccharomyces cerevisiae commercial inoculum is used to start fermentation, presented low diversity indexes (H’ = 0.7903 and 0.6442) and only S. cerevisiae, Kluyveromyces marxianus, and, sporadically, Pichia manshurica were found. Results suggest that the yeast microbiota involved in mezcal fermentation during the different seasons is affected by the type of inoculum; changes include the presence of some species that were only identified during a specific season in alcoholic fermentation, such as Torulaspora delbrueckii and Pichia kluyveri.

First Report of Lasiodiplodia pseudotheobromae Causing Postharvest Decay of Strawberries in Florida

Postharvest decay of strawberry (Fragaria × ananassa Duch.) is a major factor causing fruit losses. Strawberries were obtained from various harvests at cooling facilities located in Dover and Plant City, FL during the 2018-19 and 2019-20 seasons. After the fruits were incubated at 22ºC for up to 5 days (d) to promote disease development, Lasiodiplodia decay was observed at up to 3% from some harvests, exhibiting gray mycelia on small lesions that gradually covered the whole fruit. The fungus was isolated onto potato dextrose agar (PDA). Five isolates (SBD18-14, SBD18-277, SBD18-279, SBD19-02 and SBD19-57) were characterized. Fungal mycelia were initially grayish white and then gradually changed to gray to dark gray on PDA at 25oC, and later produced black pigments (Fig. S1). Pycnidia were observed from inoculated strawberries at 14 d. Isolates shared similar conidia morphology: aseptate, hyaline, ellipsoid to ovoid, measuring L × W: 24.0-34.0 (28.3) × 13.0-16.0 (14.3) μm (n =100). Mature conidia were brown, one septate, measuring L × W: 25.0-33.0 (28.8) × 13.0-16.0 (14.5) μm (n =100). The isolates were identified as Lasiodiplodia spp. morphologically (Alves et al. 2008). DNA was extracted from fungal mycelia using an OmniPrep DNA extraction kit, and PCR amplification of ITS and EF1-α genes was performed following the conditions described by White et al. (1990) with some modifications using primers ITS1F-F/ITS4-R (Gardes and Bruns, 1993; White et al., 1990) and EF1-668-F/EF1-1251-R (Alves et al., 2008), respectively. The BLASTn in GenBank showed that the sequences obtained had 99.61 to 100% homology with those of ITS (EF622077) and EF1-α (EF622057) from L. pseudotheobromae CBS116459 (an ex-type strain) (Alves et al., 2008). Sequences of the isolates have been deposited in GenBank with accessions OP326017 to OP326021 for ITS, and OP356202 to OP356206 for EF1-α. Phylogenetic analysis showed that these isolates clustered in the same clade (bootstrap value at 64) with L. pseudotheobromae (Fig. S2). Two fungal inoculum types (mycelia and conidia), two fruit inoculation methods (injury and non-injury) and five fungal isolates were used for pathogenicity tests. Fungal mycelia (2-day-old) on PDA plug (5 mm) or 10 µL of conidial suspension (106 spores/mL) was placed onto each injury (1 x 1 mm in size) or a non-injury area on the surfaces of five strawberry fruits (cv. Florida Brilliance). PDA plug alone or water drops placed on injury or non-injury areas on fruits served as respective controls. Inoculated and control fruits were incubated in a covered plastic container with 100% RH at 22ºC. The experiment was repeated twice. Decay initially appeared as soft and lightly discolored tissue at inoculation areas 2 d post-inoculation (dpi) that extended quickly thereafter. Brown to dark lesions on both injury- and non-injury fruits inoculated with conidia or mycelia were observed at 3 dpi. Decay and gray mycelia gradually developed over the whole fruit at 6 dpi, and pycnidia were observed after 14 dpi (Fig. S1). Disease incidence of 100% was observed on all tests. Control fruits did not develop decay. The results indicate that these isolates are pathogenic to strawberries and infect fruit via both non-injured and injured fruit surfaces. The inoculated fungal isolates were re-isolated, thus, fulfilling Koch’s postulates. L. theobromae, Neofusicoccum parvum/N. ribis species complex causing strawberry fruit rot in Florida fields was reported (Oliveira et al., 2019), but not L. pseudotheobromae. To our knowledge, this is the first report of postharvest decay caused by L. pseudotheobromae A.J.L. Phillips, A. Alves &amp; Crous on strawberries in Florida and in the USA, and it should be considered in strawberry disease management.

Perspectives and Progress in Bioethanol Processing and Social Economic Impacts

The liquid biofuel bioethanol is widely produced worldwide via fermenting sugars extracted from a variety of raw materials, including lignocellulose biomass, one of the world’s most abundant renewable resources. Due to its recalcitrant character, lignocellulose is usually pretreated by mechanical, chemical, and biological methods to maximize sugar recovery. Pretreated lignocellulose biomass undergoes a fermentation process performed sequentially or simultaneously to saccharification. The different fermentation strategies (e.g., separate or simultaneous hydrolysis and fermentation or co-fermentation) and conditions (e.g., inoculum type load, agitation, temperature, and pH) affect ethanol yield. Genetic modification of the inoculum has been focused recently to improve ethanol tolerance and as well as to use different sugars to enhance the performance of the microorganisms involved in fermentation. Nonetheless, these improvements result in a substantial increase in costs and have certain environmental costs. This review offers an overview of advancements in bioethanol production, with a primary focus on lignocellulosic feedstock, while also considering other feedstocks. Furthermore, it provides insights into the economic, social, and environmental impacts associated with bioethanol production.

Maximizing Bio-Hydrogen and Energy Yields Obtained in a Self-Fermented Anaerobic Bioreactor by Screening of Different Sewage Sludge Pretreatment Methods

Egypt faces significant challenges in managing its sewage sludge generated in large quantities from wastewater treatment plants. This study investigates the feasibility of utilizing sewage sludge as a renewable resource for hydrogen production through anaerobic digestion at the 100 L bioreactor level. Hydrogen is considered a promising alternative energy source due to its high energy content and environmental benefits. To optimize the microbial degradation process and maximize hydrogen production from sewage sludge, a specialized pretreatment is necessary. Various pretreatment methods have been applied to the sewage sludge, individually and in combination, to study the bio-hydrogen production from sewage sludge. The four methods of treatment were studied in batch assays as a pilot scale. Thermal pretreatment of sewage sludge significantly increases bio-hydrogen production yield compared to other sewage sludge pretreatment methods, producing the highest H2 yield (6.48 LH2/g VS). In general, the hydrogen yield of any type of pretreated inoculum was significantly higher than the untreated inoculum. At the same time, alkaline pretreatment improved the hydrogen yield (1.04 LH2/g VS) more than acid pretreatment (0.74 LH2/g VS), while the hydrogen yield for the combination of pretreatments (shock alkali pretreatment) was higher than both (1.73 LH2/g VS), On the other hand, untreated sewage sludge (control) had almost no hydrogen yield (0.03 LH2/g VS). The self-fermented anaerobic bioreactor improved sewage sludge utilization, increased bioenergy yields, and seems to be promising for treating complex wastes at this scale.