Continuous Production of Fumaric Acid with Immobilised Rhizopus oryzae: The Role of pH and Urea Addition

Fumaric acid is widely used in the food and beverage, pharmaceutical and polyester resin industries. Rhizopus oryzae is the most successful microorganism at excreting fumaric acid compared to all known natural and genetically modified organisms. It has previously been discovered that careful control of the glucose feed rate can eliminate the by-product formation of ethanol. Two key parameters affecting fumaric acid excretion were identified, namely the medium pH and the urea feed rate. A continuous fermentation with immobilised R. oryzae was utilised to determine the effect of these parameters. It was found that the selectivity for fumaric acid production increased at high glucose consumption rates for a pH of 4, different from the trend for pH 5 and 6, achieving a yield of 0.93 gg−1. This yield is higher than previously reported in the literature. Varying the urea feed rate to 0.255 mgL−1h−1 improved the yield of fumaric acid but experienced a lower glucose uptake rate compared to higher urea feed rates. An optimum region has been found for fumaric acid production at pH 4, a urea feed rate of 0.625 mgL−1h−1 and a glucose feed rate of 0.329 gL−1h−1.

Screening of Lactic Acid Fermentation by Rhizopus oryzae on Banana Peel Using Two-Level Factorial Analysis

For ages, pure sugars or edible crops have produced lactic acid. However, a major concern on lactic acid production lies in the cost of the raw materials used. Thus, an alternative to overcome this situation is urgently needed. Characterization of banana peels shows that it contains promising sugar that can be utilized for lactic acid production, which are xylose (0.774 g/L), glucose (0.756 g/L) and fructose (0.532 g/L). Thus, this study aims to screen the potential of banana peel as a substrate by using Rhizopus oryzae through batch fermentation for lactic acid production, as R. oryzae can synthesize lactic acid in low nutrient requirements. Two-level factorial analysis was designed to screen the effects of moisture content (60% and 80%), temperature (27 °C and 40 °C), pH (4.5 and 6.5) and inoculum size (1x104 spores/mL and 1x108 spores/g) on the lactic acid production. Based on the Two-level factorial (2LF) analysis, the highest lactic acid production of 0.0813 g/L was detected at 80 % moisture content, pH 4.5, the temperature of 27 °C and inoculum size of 1x104 spores/g. The findings show that most of the conditions have a significant difference between each other (p<0.05). Therefore, the fermentation of banana peels by R. oryzae could be a promising method to produce a lactic acid concentration.

Interrelationship Among Seed Quality Parameters and Fungi Associated with Seeds of Cotton (Gossypium Hirsutum L.)

A total of twenty nine species of fungi namely Aspergillus aculeatus Lizuka, A. flavus Link, A. fumigatus Fresenius, A. niger Van Tiegh, A. nidulans Eidam, A. subramanianii Visagie, Frisvad & Samson, A. tamariiKita G., A. toxicarius Murak, A. wentii Wehmer, Curvularial unata (Wakker) Boedijn, Colletotrichum gloeosporioides (Penz.) Sacc., C. gossypii Southw., Chaetomium globosum Kunze., Fusarium moniliforme J. Shelden, F. nivale (Fr.) Sorauer, F. oxysporum Schlechtendal, F. fujikuroi Nirenberg, F. solani(Mart.) Sacc., Lasiodiplodiatheobromae (Pat.) Griffon & Maubl., Meyerozyma guilliermondii (Wick.) Kurtzman & M. Suzuki., Mucor sp. P. Micheli ex L., Penicillium aculeatum Raper& Fennell, Penicillium citrinum Thom, Rhizoctonia solani Khun., Rhizopus stolonifer (Ehrenb.) Vuill., Rhizopus oryzae Went & Prins. Geerl., Rhizo mucor sp. Luce t & Costantin, Syncephalastrum racemosum Cohn and Trichoderma viride Pers. were found to be associated with the seeds of 14 varieties viz. CB-1 to CB-14 of cotton. Seed quality analysis showed that percentage of pure seeds, germination, moisture, seedling mortality and associated fungi with different varieties of cotton seeds varies from 97.08-99.92%, 80-93%, 10-11.3%, 16.05-50.30% and 24-78.0% respectively. Present study deals with estimation of interrelationship between some quality factors through correlation and regression analysis are important for the assessment of seed quality. There were negative correlations between seedling mortality and purity percentage and between germination rate and fungus frequency. On the other hand, positive correlations were found between germination rate and purity percentage, between seedling mortality and fungus frequency, between purity percentage and fungus frequency, between germination rate and seed moisture and between fungus frequency and seed moisture. CB 8 was superior from the other cotton variety as it showed higher physical purity of seed (98.41%), higher seed germination (92%), lower fungal incidence (24%) and lower mortality of seedling (23.91%). Bioresearch Commu. 8(1): 1061-1067, 2022 (January)

Evaluation of Antimicrobial and Anticancer Activities of Selected Medicinal Plants of Himalayas, Pakistan

Medicinal plants are known for their diverse use in the traditional medicine of the Himalayan region of Pakistan. The present study is designed to investigate the anticancer and antimicrobial activities of Prunus cornuta and Quercus semicarpifolia. The anticancer activity was performed using cancerous human cell lines (HepG2, Caco-2, A549, MDA-MB-231, and NCI-H1437 carcinoma cells), while the antimicrobial activity was conducted with the agar-well diffusion method. Furthermore, toxicity studies were performed on alveolar and renal primary epithelial cells. Initially, different extracts were prepared by maceration techniques using n-hexane, chloroform, ethyl acetate, butanol, and methanol. The preliminary phytochemical screening showed the presence of secondary metabolites such as alkaloids, tannins, saponins, flavonoids, glycosides, and quinones. The chloroform extract of P. cornuta (PCC) exhibited significant inhibitory activity against Acinetobacter baumannii (16 mm) and Salmonella enterica (14.5 mm). The A. baumannii and S. enterica strains appeared highly susceptible to n-hexane extract of P. cornuta (PCN) with an antibacterial effect of 15 mm and 15.5 mm, respectively. The results also showed that the methanolic extracts of Quercus semecarpifolia (QSM) exhibited considerable antibacterial inhibitory activity in A. baumannii (18 mm), Escherichia coli (15 mm). The QSN and QSE extracts also showed good inhibition in A. baumannii with a 16 mm zone of inhibition. The Rhizopus oryzae strain has shown remarkable mycelial inhibition by PCM and QSN with 16 mm and 21 mm inhibition, respectively. Furthermore, the extracts of P. cornuta and Q. semicarpifolia exhibited prominent growth inhibition of breast (MDA-MB-231) and lung (A549) carcinoma cells with 19–30% and 22–39% cell viabilities, respectively. The gut cell line survival was also significantly inhibited by Q. semicarpifolia (24–34%). The findings of this study provide valuable information for the future development of new antibacterial and anticancer medicinal agents from P. cornuta and Q. semicarpifolia extracts.

First Report of Aspergillus flavus Causing Fruit Rot on Kiwifruit in China

In October 2020, fruit rot symptoms were detected on kiwifruit (Actinidia chinensis var. deliciosa ‘Xuxiang’) in southwestern Shaanxi (Hanzhong municipality; 107.27° E, 33.23° N) in China. Mature kiwifruit, during the harvest period, exhibited soft rot and brown lesions. The symptoms were similar than those reported for Alternaria alternata, Colletotrichum spp., Fusarium avenaceum and Rhizopus oryzae causing fruit rot on kiwifruit (Feng et al. 2019; Li et al. 2017; Kim et al. 2018; Zhao et al. 2020). The symptoms were observed in approximately 15% of the fruit in 6 kiwifruit orchards (31 ha in total). Ten samples of symptomatic tissue, approximately 1 cm2 in size, were sterilized in 2% NaOCl for 30 seconds and washed twice with sterilized water. The pathogen was isolated from all collected samples via culturing on PDA medium, containing 50 µg/mL chloramphenicol, at 28 ºC. Green powdery-like colonies were detected after 5 days (Figure 1). A total of 12 isolates were obtained via single spore isolation. Internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and RNA polymerase II subunit (RPB2) genes were amplified using ITS5/ITS4, A_EF1_F/A_EF1_R and RPB2-5F/RPB2-7cR (NJC03), or RPB2-7cF/RPB2-11aR (NJC04), primers, respectively. Eleven isolates shared the same sequences (NJC03), MZ801787 (ITS), MZ701709 (EF1-α) and MZ701707 (RPB2), while one of the isolates provided different sequences (NJC04), OK618459 (ITS), OK634020 (EF1-α) and OL331017 (RPB2). The obtained ITS sequences shared >99% homology to the ITS gene from A. flavus KU20018.4 (MT487825), the EF1-α sequences shared 100% homology to the EF1-α gene from A. flavus clinical2342 (KP054370) and the RPB2 sequences shared >99% homology to the RPB2 genes from A. flavus PW3170 (LC000581) and A. flavus NRRL3357 (XM_041293948). Molecular phylogenetic tree was constructed using MEGA7 with reference Aspergillus strains (Figure 2). Microscope observations of all isolates showed the presence of septate mycelium, circular unicellular conidia (2-4 µm diameter) and conidiophores, and agree with the morphology of A. flavus (Horn 2005). The pathogenicity of all isolates was screened using intact and wounded ‘Xuxiang’ kiwifruits (ten kiwifruits were used for each combination with 3 replicates), which were purchased from a local market. A 1 × 106 spores/mL (10 µL) solution of the isolates was used for the inoculation. Sterilized water was used in the control experiment. Inoculated kiwifruits were storage at 26 °C and 60% relative humidity for 10 days. Rot lesions in the wounded kiwifruits were totally covered by green mycelia, while the lesions on the intact kiwifruits were similar to the symptoms observed in the field. The pathogen was recovered and its identity was confirmed by sequence analysis of ITS, EF1-α and RPB2, fulfilling Koch’s postulates. A. flavus is known to be an important fungal pathogen of corn, cotton and peanuts (Zhang et al. 2020). During recent years, A. flavus was reported to cause fruit rot on grapes (Ghuffar et al. 2020), and was identified on almond, fig, organic spelt and pistachio (Krulj et al. 2017; Ortega-Beltran et al. 2019). The presence of A. flavus in food products is an issue of global concern due to A. flavus is able to produce carcinogenic aflatoxin (Maxwell et al. 2021). As far as we know, this is the first report of A. flavus causing fruit rot on kiwifruit. This report will help to understand the distribution of A. flavus in crops and the food safety hazards that are present in China.

Transcriptome analysis of Rhizopus oryzae seed pellet formation using triethanolamine

Rhizopus oryzae (R. oryzae) can effectively produce organic acids, and its pellet formation in seed cultures has been shown to significantly enhance subsequent fermentation processes. Despite advances in strain development, simple and effective methods for inducing pellet morphology and a basic understanding of the mechanisms controlling this process could facilitate substantial increases in efficiency and product output. Here, we report that 1.5% triethanolamine (TEOA) in seed culture medium can activate the growth of R. oryzae spores in compact and uniform pellets which is optimal for fermentation conditions. Analysis of fermentation kinetics showed that the production of fumaric and L-malic acid increases 293% and 177%, respectively. Transcriptomic analysis revealed that exposure of R. oryzae to 1.5% TEOA during the seed culture activated the phosphatidylinositol and mitogen-activated protein kinase signaling pathways. Theses pathways subsequently stimulated the downstream carbohydrate-active synthases and hydrolases that required for cell wall component synthesis and reconstruction. Our results thus provide insight into the regulatory pathways controlling pellet morphology germane to the viability of seed cultures, and provide valuable reference data for subsequent optimization of organic acid fermentation by R. oryzae.

Degradation of Lignocelluloses Cocoa Shell (Theobroma cacao L.) by Various Types of Mould Treatments

Lignocellulose can be degraded by lignocellulolytic microorganisms such as moulds. The purpose of the study was to obtain the right type of moulds in degrading lignocellulose on the cocoa shell powder. The study used a completely randomized design method using four treatments of different types of mould (Trichoderma viride, Neurospora sitophila, Aspergillus niger, and Rhizopus oryzae) towards cocoa shell powder fermentation. Solid fermentation of cocoa shell powder was carried out for 5 days in an incubator with a temperature of 30°C for T. viride, N. sitophila, and R. oryzae, while A. niger of 35°C. The fermented substrate was then dried in a cabinet oven with a temperature of 50°C for 4 days. Tests of lignin, cellulose, and hemicellulose were performed towards the treatments by the Chesson method, while the moisture content test was performed using the AOAC method. Degradation of fermented cocoa shell powder has shown a significant effect on moisture, lignin, cellulose, and hemicellulose contents. Trichoderma viride resulted in the highest lignocellulose degradation compared with the other treatments. The percentage decrease of lignin content is up to 46.69 wt%; while cellulose of 22.59 wt%; and hemicellulose is about 19.41 wt% from the initial lignin weight.