Liquid Culture Production Research Articles

Production, physico-chemical properties and antioxidant activity of melanin from Annulohypoxylon stygium (Lév.) Y.M. Ju, J.D. Rogers and H.M. Hsieh

To obtain higher melanin production in liquid culture, culture conditions of Annulohypoxylon stygium (Lév.) Y.M. Ju, J.D. Rogers and H.M. Hsieh were optimised. The results showed that using single factor experiment and orthogonal test, the optimised production of melanin reached 2.20 g/L, which was 2.06 times higher than that of the control group. In addition, it was speculated that A. stygium melanin (AsM) was 3,4-dihydroxyphenylalanine (DOPA) melanin and showed an amorphous irregular structure. Moreover, it had good solubility in alkaline solution. AsM showed good antioxidant activity at a concentration of 500 mg/L, with DPPH, ABTS and OH radicals scavenging activities of 90.83%, 75.36% and 70.90%, respectively. AsM prevented alcohol-induced oxidative damage and oxidative stress in HepG2 cells by inhibiting the decrease of antioxidant key enzyme activity under alcohol stimulation. It was proved to have a great potential for application as a natural antioxidant and a substitute for synthetic pigments.

In vitro liquid culture production and post-production pathogenicity of the hybrid Heterorhabditis bacteriophora HBH strain

Entomopathogenic nematodes (EPNs) are biological control agents that can be used as alternatives to chemical pesticides against insect pests. Liquid culture is the most suitable method for large-scale production of EPNs. However, to achieve commercial success, the optimization of production parameters is crucial. This study aimed to identify the optimal temperature, pH and dissolved carbon dioxide concentration for the liquid culture production of Heterorhabditis bacteriophora hybrid HBH strain. Temperature and pH experiments were conducted in Erlenmeyer flasks, while carbon dioxide experiments were conducted using a 5 L bench-top fermenter. The results revealed that the best yield was achieved at a temperature of 26 °C, a pH of 9, and a dissolved carbon dioxide concentration of 8–9%. The highest yield was observed between the 14th to 16th days in almost all batches. Although no significant difference was found in the pathogenicity of the populations, certain populations obtained from carbon dioxide trials exhibited greater efficacy at lower doses than in vivo populations. The liquid production of the HBH hybrid strain is a critical step towards commercial production. Further goals include the optimization of additional parameters for large-scale production. The findings of this study are expected to have practical implications for the industrialization of native hybrid EPN strains.

Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A.luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

Impact of egg yolk type in liquid culture production of Steinernema carpocapsae JAP1 and their virulence

Dehydrated egg yolk is usually incorporated as a powder into the liquid medium to supply protein for entomopathogenic nematodes (EPNs) culture. This study aimed to evaluate the yield production of Steinernema carpocapsae strain JAP1 cultured in 10 mL of the P2 medium with fresh liquid egg-yolk (P2-LEY) and powdered egg-yolk (P2-PEY) into orbitally shaken bottles at 130 rpm and 28 °C for 16 days. The nematode inoculum was transferred from the surface layer of a solid culture with 21.5 % of infective juveniles (IJs), to achieve an initial concentration of 2000 ± 200 IJs·mL−1. Using P2-LEY, the maximum concentration was improved to 265 000 nematodes·mL−1 with 60% of the population in the IJ stage. In addition, the mortality of Galleria mellonella due to confrontation with these juveniles was 61%, which shows a non-statistical difference with the virulence of IJs in vivo reared in host insects. We concluded that LEY is a suitable component for the liquid culture mass production of nematodes. These results are discussed in light of information on the nutritional differences between LEY and PEY and their emulsifier attributes.

INDOLE ACETIC ACID AND EXOPOLYSACCHARIDE PRODUCTION OF NITROGEN FIXING BACTERIA ISOLATED FROM NUTMEG TREE

The constraint of plant cultivation in the tropics is low nitrogen (N) content in soil. The application of nitrogen fixing bacteria (NFB) is not only fulfill the N demand but also provide the phytohormones Indole acetic acid (IAA) and exopolysaccharides (EPS). The objective of the experiment was to was to isolate the NFB from the rhizosphere of nutmeg tree and characterized their IAA and EPS production in a liquid culture. The NFB was isolated by serial dilution plate method on Tryptic Soy Agar. In order to test the ability of various NFB isolates to produce IAA and EPS, all isolates were grown in Tryptic Soy Broth for 72 hours at room temperature. The results showed that at the end of experiment the media acidity was shift from neutral to alkaline. The population of five isolates of NFB was approximately 8-9 log10 (of CFU/mL). The IAA production in liquid culture of all isolates was similar but the EPS production was varied and depend on the isolates, The highest EPS content was in PL1 culture and the lowest was in the culture of PL5. This experiment verified that the NFB isolated from nutmeg rhizosphere enable to produce IAA and EPS.

Phosphoproteome Dynamics of Streptomyces rimosus during Submerged Growth and Antibiotic Production.

ABSTRACTStreptomyces rimosus is an industrial streptomycete, best known as a producer of oxytetracycline, one of the most widely used antibiotics. Despite the significant contribution of Streptomyces species to the pharmaceutical industry, most omics analyses have only been conducted on the model organism Streptomyces coelicolor. In recent years, protein phosphorylation on serine, threonine, and tyrosine (Ser, Thr, and Tyr, respectively) has been shown to play a crucial role in the regulation of numerous cellular processes, including metabolic changes leading to antibiotic production and morphological changes. In this study, we performed a comprehensive quantitative (phospho)proteomic analysis during the growth of S. rimosus under conditions of oxytetracycline production and pellet fragmentation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis combined with phosphopeptide enrichment detected a total of 3,725 proteins, corresponding to 45.6% of the proteome and 417 phosphorylation sites from 230 phosphoproteins. Significant changes in abundance during three distinct growth phases were determined for 494 proteins and 98 phosphorylation sites. Functional analysis revealed changes in phosphorylation events of proteins involved in important cellular processes, including regulatory mechanisms, primary and secondary metabolism, cell division, and stress response. About 80% of the phosphoproteins detected during submerged growth of S. rimosus have not yet been reported in streptomycetes, and 55 phosphoproteins were not reported in any prokaryote studied so far. This enabled the creation of a unique resource that provides novel insights into the dynamics of (phospho)proteins and reveals many potential regulatory events during antibiotic production in liquid culture of an industrially important bacterium.IMPORTANCE Streptomyces rimosus is best known as a primary source of oxytetracycline (OTC). The significant global market value of OTC highlights the need for a better understanding of the regulatory mechanisms that lead to production of this antibiotic. Our study provides, for the first time, a detailed insight into the dynamics of (phospho)proteomic profiles during growth and antibiotic production in liquid culture of S. rimosus. Significant changes in protein synthesis and phosphorylation have been revealed for a number of important cellular proteins during the growth stages that coincide with OTC production and morphological changes of this industrially important bacterium. Most of these proteins have not been detected in previous studies. Therefore, our results significantly expand the insight into phosphorylation events associated with important cellular processes and antibiotic production; they also greatly increase the phosphoproteome of streptomycetes and contribute with newly discovered phosphoproteins to the database of prokaryotic phosphoproteomes. This can consequently lead to the design of novel research directions in elucidation of the complex regulatory network in Streptomyces.

Comparing Production and Efficacy of Cordyceps javanica With Cordyceps fumosorosea.

A newly discovered entomopathogenic fungus Cordyceps javanica (Friedrichs & Bally) Samson & Hywel-Jones (Hypocreales: Cordycipitaceae) strain Wf GA17 was compared with the commercial Cordyceps fumosorosea Wize (Hypocreales: Cordycipitaceae) Apopka 97 strain for liquid-culture production, formulation, insecticidal efficacy, and storage stability under laboratory conditions. We compared culture media with carbon:nitrogen (C:N) ratios of 10:1, 30:1, and 50:1 for these two isolates. A third strain, C. fumosorosea strain ARSEF 3581, had previously been optimized for liquid-culture production of blastospores at 10:1 C:N served as an added control. These seven cultures were processed by spray drying with skim milk powder, stored at 25oC to evaluate storage stability, and assayed for insecticidal activity against Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) neonates. Final blastospore concentrations were not significantly different among cultures, ranging from 4.47 to 9.88 × 108 spores/ml. Fungal biomass decreased and final glucose concentrations increased with increasing C:N ratios, indicating better fungal growth with higher nitrogen concentrations. Product yields from the spray dryer (grams per liter culture) increased with increased C:N ratios while spore concentrations decreased, ranging from 2.27 to 7.17 × 109 spores/g. There were no significant differences for insecticidal efficacy among the seven treatments. Spores produced in 10:1 C:N ratio media retained viability longer than spores produced in other media. Cost of ingredients decreased with increasing C:N ratios, such that the 30:1 media may yield the most economical product. The raw material cost needed for application was 1.4× greater for Wf GA17 compared with Apopka 97, a difference that could be erased by optimization of culture conditions.

New role of a histone chaperone, HirA: Involvement in kojic acid production associated with culture conditions in Aspergillusoryzae

Kojic acid (KA) is a representative secondary metabolite of Aspergillus oryzae, but the underlying molecular mechanisms that regulate KA production are unknown. This study tried to find a genetic factor of KA production in A.oryzae, with a special focus on liquid cultures. We screened a gene predicted to encode HirA, a subunit of the histone chaperon, the HIR complex. A gene disruption strain of hirA showed decreased KA production in liquid culture, whereas it showed increased KA production in plate culture. We confirmed that a decrease/increase of KA production observed by hirA disruption was caused by altered expression of kojA and kojR. These observations suggested the regulatory role of histone chaperon in secondary metabolism in filamentous fungi. So far as we know, this report is the first showing that disruption of a gene resulted in the opposite effect on KA production in liquid and plate cultures in A.oryzae.

Enhancement of the Aroma Compound 2-Acetyl-1-pyrroline in Thai Jasmine Rice (Oryza sativa) by Rhizobacteria under Salt Stress.

Simple SummaryThe major aroma compound (2-acetyl-1-pyrroline) of the world-famous Thai jasmine rice, variety KDML105, has declined due to high soil salinity and agrochemical input. In this work, the rhizobacteria from rice were investigated for the aroma compound’s production, as well as their potential for increasing the compound content in Thai jasmine rice seedlings under saline conditions. Our results provide evidence that the addition of aroma compound-producing rhizobacteria increases the aroma content in the rice seedlings under salt stress. Sinomonas sp. strain ORF15-23 which colonize the rice roots, is a promising rhizobacteria in promoting the aroma level of the Thai jasmine rice grown under salt stress and could be developed as a bioinoculant for Thai jasmine rice cultivation in a salt-affected area.Thai jasmine rice (Oryza sativa L. KDML105), particularly from inland salt-affected areas in Thailand, is both domestically and globally valued for its unique aroma and high grain quality. The key aroma compound, 2-acetyl-1-pyrroline (2AP), has undergone a gradual degradation due to anthropogenic soil salinization driven by excessive chemical input and climate change. Here, we propose a cheaper and an ecofriendly solution to improve the 2AP levels, based on the application of plant growth-promoting rhizobacteria (PGPR). In the present study, nine PGPR isolates from rice rhizosphere were investigated for the 2AP production in liquid culture and the promotion potential for 2AP content in KDML105 rice seedlings under four NaCl concentrations (0, 50, 100, and 150 mM NaCl). The inoculation of 2AP-producing rhizobacteria resulted in an increase in 2AP content in rice seedling leaves with the maximum enhancement from Sinomonas sp. ORF15-23 at 50 mM NaCl (19.6 µg·kg−1), corresponding to a 90.2% increase as compared to the control. Scanning electron microscopy confirmed the colonization of Sinomonas sp. ORF15-23 in the roots of salinity-stressed KDML105 seedlings. Our results provide evidence that Sinomonas sp. ORF15-23 could be a promising PGPR isolate in promoting aroma level of Thai jasmine rice KDML105 under salt stress.

Production of entomopathogenic nematodes in submerged monoxenic culture: A review.

Monoxenic liquid culture is the most suitable technology for scaling up to industrial production of entomopathogenic nematodes (EPNs); however, the variability of the yield production remains a current problem in the process. The aim of this study was to analyze the parameters and criteria for EPN production in liquid culture based on scientific and technological knowledge from the last two decades. While experimental research has permitted the yield production of Heterorhabditis bacteriophora (362 × 103 infective juveniles [IJs]/ml) and Steinernema carpocapsae (252 × 103 IJs/ml), simultaneously, theoretical approaches have contributed to the understanding of the culture process, based on biological parameters of the bacterium-nematode complex and hydrodynamic and rheological parameters of the complex gas-liquid-solid system. Under this interdisciplinary research approach, bioprocess and biosystem engineering can contribute to design the various control strategies of the process variables, increase the productivity, and reduce the variability that until now distinguishes the in vitro production of EPNs by the liquid culture.

In vitro liquid culture and optimization of Steinernema jeffreyense using shake flasks

Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml−1 after 14 days, in comparison to 75% and 99,875 IJs ml−1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml−1), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.

Ammonium Acetate Supplement Strategy for Enhancement of Chaetominine Production in Liquid Culture of Marine-Derived Aspergillus fumigatus CY018.

Pharmacological research on (CHA), a marine-derived quinazolinone alkaloid with significant cytotoxic activity, is restricted by low yields and is a problem that needs to be settled urgently. In this work, the selection of additional nitrogen sources and the optimization of additional concentrations and longer fermentation times using ammonium acetate, were investigated. CHA production was optimized to 62.1 mg/l with the addition of 50 mM ammonium acetate at 120 h of the fermentation in the shaker flask. This feeding strategy significantly increased 3- deoxy-arabino-heptulosonate-7-phosphate synthase activity and transcript levels of critical genes (laeA, dahp and trpC) in the shikimate pathway compared with the non-treatment group. In addition, the selection of the feeding rate (0.01 and 0.03 g/l/h) was investigated in a 5-L bioreactor. As a result, CHA production was increased by 57.9 mg/l with a 0.01 g/l/h ammonium acetate feeding rate. This work shows that the strategy of ammonium acetate supplementation had an effective role in improving CHA production by Aspergillus fumigatus CY018. It also shows that this strategy could serve as an important example of large-scale fermentation of a marine fungus in submerged culture.

Identification of amino acids negatively affecting Fusarium trichothecene biosynthesis.

Nitrogen sources in media have a significant impact on the onset of secondary metabolism in fungi. For transcriptional activation of many nitrogen catabolic genes, an AreA transcription factor is indispensable. This also holds true for Fusarium graminearum that produces trichothecenes, an important group of mycotoxin, in axenic culture. Despite the presence of numerous consensus AreA-binding sites in the promoters of Tri genes in the trichothecene cluster core region, the effect of medium amino acids on trichothecene biosynthesis is poorly understood. In this study, we examined the effect of certain amino acids, which were predicted to activate AreA function and increase Tri gene transcription, on trichothecene production in liquid culture. By frequent monitoring and adjustments in the pH of the culture medium, including replacement of the spent medium with fresh medium, we demonstrate the suppressive effects of the amino acids, used as the sole nitrogen source, on trichothecene biosynthesis. When the medium pH was maintained at 4.0, Gly, L-Ser, and L-Thr suppressed trichothecene production by F. graminearum. Enhanced trichothecene-inducing effects were observed when the medium pH was 3.5, with only L-Thr suppressing trichothecene synthesis.

Engineering Escherichia coli to increase triacetic acid lactone (TAL) production using an optimized TAL sensor-reporter system.

Triacetic acid lactone (TAL) (4-hydroxy-6-methyl-2-pyrone) can be upgraded into a variety of higher-value products, and has potential to be developed into a renewable platform chemical through metabolic engineering. We previously developed an endogenous TAL sensor based on the regulatory protein AraC, and applied it to screen 2-pyrone synthase (2-PS) variant libraries in E. coli, resulting in the identification of variants conferring up to 20-fold improved TAL production in liquid culture. In this study, the sensor-reporter system was further optimized and used to further improve TAL production from recombinant E. coli, this time by screening a genomic overexpression library. We identified new and unpredictable gene targets (betT, ompN, and pykA), whose plasmid-based expression improved TAL yield (mg/L/OD595) up to 49% over the control strain. This work further demonstrates the utility of customized transcription factors as molecular reporters in high-throughput engineering of biocatalytic strains.

Inhibition of Fusarium trichothecene biosynthesis by yeast extract components extractable with ethyl acetate

While Fusarium graminearum readily produces trichothecenes in complex media containing sucrose as the carbon source (YS_60), the amount of the mycotoxin is quite limited when other sugars, such as glucose and fructose, are used. We found that autoclaving of media containing fructose and yeast extract (YF_60) results in the formation of inhibitors of trichothecene biosynthesis by F. graminearum JCM 9873, a strain that produces 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Removal of the solvent fraction from the autoclaved media after ethyl acetate extraction attenuated the inhibitory activity against trichothecene production. In addition, extraction of the non-autoclaved complex media with ethyl acetate, followed by removal of the solvent fraction, similarly resulted in increased accumulation of the mycotoxin. Although the increase in trichothecene production differed considerably among fungal strains and yeast extract products, F. graminearum species complex generally responded to the medium treatments in the same way. These results suggest that some hydrophobic substances that arise during the drying and heating of yeast extract negatively affected trichothecene production in liquid culture. Modes of actions of inhibitory substances were partially characterized using strain JCM 9873, with focus on the transcriptional and functional analyses of Tri6, a key regulator gene in trichothecene biosynthesis. The presence of the ethyl acetate-extractable substances in autoclaved YF_60 media decreased the relative transcription level of Tri6, as well as that of a trichodiene synthase gene Tri5. Thus, the substances exerted their inhibitory action through suppression of Tri6 expression. By using a yeast extract lot that completely prevented trichothecene production by the wild-type strain in autoclaved YS_60 medium, we prepared YF_60 media and cultured a constitutive Tri6 overexpressor strain described by Maeda et al. (2018). Despite the high transcription level of Tri6, the presence of the ethyl acetate extractable-substances suppressed 15-ADON production. These results suggested that both Tri6p-independent initial activation of Tri6 expression and subsequent Tri6p-dependent activation of Tri expression were affected by the hydrophobic substances in the yeast extract products.

Characterization, culture medium optimization and antioxidant activity of an endophytic vitexin-producing fungus Dichotomopilus funicola Y3 from pigeon pea [Cajanus cajan (L.) Millsp.

The aim of this study was to characterize a fungal endophyte Y3 from pigeon pea (Cajanus cajan [L.] Millsp), as a novel producer of vitexin, and its culture medium optimization and antioxidant activity. The endophyte from the leaves of pigeon pea was identified as Dichotomopilus funicola by the morphological and molecular characteristics. The most important medium variables affecting vitexin production in liquid culture of D. funicola Y3 were screened by Plackett-Burman design, and three culture medium constituents (i.e. l-phenylalanine, salicylic acid and CuSO4 ·5H2 O) were identified to play significant roles in vitexin production. The most significant factors were further optimized using by central composite design with response surface methodology. The DPPH radical-scavenging assay indicated that fungal vitexin exhibited notable antioxidant activity with an EC50 value of 164μgl-1 . First, a novel endophyte vitexin-producing Dichotomopilus funicola Y3 was isolated from pigeon pea (Cajanus cajan[L.] Millsp.). The maximum vitexin yield was obtained as78·86mgl-1 under the optimum culture medium constituents: 0·06gl-1 l-phenylalanine, 0·21gl-1 salicylic acid, and 0·19gl-1 CuSO4 ·5H2 O in medium, which is 4·59-fold higher than that in the unoptimized medium. Also, fungal vitexin clearly demonstrated its antioxidant potential. These findings provide an alternative source for large-scale production of vitexin by endophytic fungal fermentation and have a promising prospect in food and pharmaceutical industry.

Functional Analysis of Aspergillus nidulans Genes Selected by Proteomic Analysis under Conditions Inducing Asexual Development

Aspergillus nidulans의 포자형성 및 무성분화에 관여하는 새로운 인자를 찾고자, 포자형성 촉진 조건인 0.6 M KCl이 첨가된 배지와 첨가되지 않은 배지에서 자란 균사체의 단백질체 분석을 시도하였다. 2DE 분석을 통해 2,400여개의 spot을 확인하였고, 무성분화 유도 9시간 및18시간 별로 생성양의 변화양상을 기준으로 총 5개의 그룹으로 나눌 수 있었다. 기능 분석이 아직까지 이루어지지 않은 단백질을 암호화하고 있는 유전자들 가운데 DU 그룹에 속하는 AN1342와 DD 그룹에 속하는 AN9419 두 개의 유전자의 기능을 알아보고자 유전자결손 돌연변이주를 제작하고 표현형을 관찰하였다. Alanine-glyoxylate aminotransferase의 기능을 할 것으로 예측되는 AN1342을 결손 시키면 무성분화 기관인 stalk의 길이가 짧아졌고, 액체 배양 시 야생형과 달리 배지로 분홍색 색소가 분비되어 sspA라 명명하였다. AN9419 결손균주는 균사생장이 심각하게 저해되고 알라닌이 첨가되어야 균사생장이 가능하였으나 무성포자 형성은 거의 이루어지지 않았으며 GO분석을 통하여 alanyl-tRNA synthetase의 기능을 할 것으로 유추된 점에 근거하여 alaA라 명명하였다. Despite the significance of external environmental factors in differentiation, putative factors involved in differentiation of Aspergillus nidulans have not yet been fully understood. A sporulation-specific proteome analysis of A. nidulans in the present study revealed that the expression levels of more than 2,400 proteins were affected under conditions inducing sporulation (0.6 M KCl) compared with normal conditions. Among the proteins with predicted functions, two targets, AN1342 and AN9419, were functionally analyzed using targeted deletion strains and phenotypic observations. For AN1342, because the deletion of the corresponding open reading frame caused a reduction in stalk length during asexual development and in pigment production in liquid culture, the gene was designated as sspA (<TEX>$\underline{s}hort$</TEX> <TEX>$\underline{s}talk$</TEX> & <TEX>$\underline{p}igment$</TEX>). Deletion of the AN9419 gene, which is predicted to encode alanyl-tRNA synthetase, led to severe growth defects due to alanine auxotrophy and abolishment of asexual reproduction and thus, the gene was designated as alaA.

Enhancement of botrallin and TMC-264 production in liquid culture of endophytic fungus Hyalodendriella sp. Ponipodef12 after treatments with metal ions

Background: Hyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions ( i.e. , Mn 2 + , Na + , Mg 2 + , Zn 2 + , Cu 2 + , Fe 2 + , Fe 3 + and Al 3 + ) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated. Results: Three most effective metal ions (Zn 2 + , Cu 2 + and Mg 2 + ) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn 2 + , Cu 2 + and Mg 2 + were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of Zn 2 + , Cu 2 + and Mg 2 + on the production of botrallin and TMC-264 by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) were evaluated, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn 2 + at 0.81 mmol/L, Cu 2 + at 0.20 mmol/L, and Mg 2 + at 0.13 mmol/L in medium. Conclusion: The results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy. 800x600 800x600 Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif;} Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif;}

Isolation of Trichoderma Spp. from Desert Soil, Biocontrol Potential Evaluation and Liquid Culture Production of Conidia Using Agricultural Fertilizers

Isolation of Trichoderma Spp. from Desert Soil, Biocontrol Potential Evaluation and Liquid Culture Production of Conidia Using Agricultural Fertilizers

Isolation of Trichoderma Spp. from Desert Soil, Biocontrol Potential Evaluation and Liquid Culture Production of Conidia Using Agricultural Fertilizers

Three Trichoderma isolates were obtained from sandy soils collected at the “Gran Desierto de Altar” in the northwest of Mexico and characterized by morphologic and molecular analyses as Trichoderma harzianum 8.4, Trichoderma asperellum 12-2 and Trichoderma asperellum BP60. Isolate T. asperellum BP60 inhibited Setophoma terrestris, grew above 50°C, and produced chitinases and siderophores, therefore it was chosen to obtain enough biomass and conidia for field applications. Conidia production was intended in liquid culture fermentation using food grade ingredients and agricultural fertilizers. Assays were done using baffled Erlenmeyer flasks containing 75 mL of culture media, kept under constant agitation at 150 RPM, with initial pH adjusted to 6.5 (NaOH 1N) at 28 ± 2°C and evaluated at 3, 6, 9 and 12 days after inoculation (DAI). Among the carbon sources, sucrose and vinaze; the former induced higher yields of biomass and conidia. Regarding nitrogen sources, the fertilizer (NH4) NO3 induced higher conidia yield. V8 juice (V8) induced the highest effect on production of both biomass and conidia. Therefore, maximum yield was 1.06 × 109 conidia.mL-1, with the formulation with 5 g of KH2PO4 (MKP, Greenhow®), 1.3 g of MgSO4•7H2O (Sul-Mag, Penoles®), 20 mg of FeCl3•6H2O (Fermont®), 150 ml of V8, 10 ml of vinaze and 2.5 g.L-1 of (NH4)NO3. Results presented here prove the potential for using an alternative, low cost, liquid media to produce conidia of T. asperellum.