Expression of genes encoding cellulolytic enzymes in some Aspergillus species.

Fermentation is an important industrial process for microbial metabolite development and has wide applications in various fields. Aspergillus is the most important genus of fungi used for the production of microbial enzymes such as cellulase. The Aspergillus genome encodes various cellulolytic enzymes. In this study, we assayed the gene expression and cellulolytic enzyme production of three isolates: A. niger (KSU009), A. terreus (KC462061), and A. flavus (KSU014). Two fermentation systems, submerged fermentation and biofilm fermentation (BF), were used for this purpose. Gene expression analysis by RT-PCR showed that cbhB, exo, eglA, eglB, eglC, and ß-actin genes were differentially expressed in the two fermentation systems for these three isolates during enzyme production. Furthermore, the expression of all genes was found to be higher in the BF system. The six genes were not expressed in the isolates with no cellulolytic enzyme production. The isolates were identified by morphological and molecular methods, which were based on macroscopic characteristics and sequence analysis of ITS1, ITS2, and the 5.8S regions of rDNA.

Molecular characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates collected from corn grains.

Twelve species from six fungal genera were found to be associated with corn (Zea mays L.) grain samples collected from three main regions of Saudi Arabia. The average frequencies of the most common genera were Aspergillus (11.4%), Fusarium (9.5%), Penicillium (5.1%), and Alternaria (5.8%). Fifteen isolates of Aspergillus flavus were screened by HPLC for their ability to produce aflatoxins (AF). The percentage of aflatoxigenic A. flavus isolates was 53%. Eight isolates produced AF, at concentrations ranging 0.7-2.9 ppb. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers were used to genetically characterize isolates of A. flavus and to discriminate between the aflatoxigenic and non-aflatoxigenic isolates. RAPD and ISSR analysis revealed a high level of genetic diversity in the A. flavus population, which was useful for genetic characterization. The clustering in the RAPD and ISSR dendrograms obtained was unrelated to geographic origin. The RAPD and ISSR markers could not discriminate between aflatoxigenic and non-aflatoxigenic isolates, but the ISSR primers were somewhat better.

Molecular characterization of Aspergillus flavus and aflatoxin contamination of wheat grains from Saudi Arabia.

Twelve species belonging to six fungal genera were found to be associated with wheat (Triticum aestivum L.) grain samples collected from three main regions in Saudi Arabia. The most common genera (average frequency) were Aspergillus (14.3%), Fusarium (29.1%), Penicillium (9.3%), and Alternaria (8.2%). Nineteen isolates of Aspergillus flavus were screened for their ability to produce aflatoxins using HPLC. Thirteen isolates produced aflatoxins ranging from 0.5 to 2.6 µg/kg. Inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of A. flavus to discriminate between aflatoxigenic and non-aflatoxigenic isolates. RAPD and ISSR analysis revealed a high level of genetic diversity in the A. flavus population, useful for genetic characterization. Clustering based on RAPD and ISSR dendograms was unrelated to geographic origin. RAPD and ISSR markers were not suitable to discriminate aflatoxigenic and non-aflatoxigenic isolates, but ISSR primers were better compared to RAPD.

Molecular characterization of the pathogenic plant fungus Rhizoctonia solani (Ceratobasidiaceae) isolated from Egypt based on protein and PCR-RAPD profiles.

Twenty-one isolates of Rhizoctonia solani were categorized into three anastomosis groups consisting of AG-4-HG-I (eight isolates), AG-2-2 (nine isolates) and AG-5 (four isolates). Their pathogenic capacities were tested on cotton cultivar Giza 86. Pre-emergence damping-off varied in response to the different isolates; however, the differences were not significant. Soluble proteins of the fungal isolates were electrophoresed using SDS-PAGE and gel electrophoreses. A dendrogram of the protein banding patterns by the UPGMA of arithmetic means placed the fungal isolates into distinct groups. There was no evidence of a relationship between protein dendrogram, anastomosis grouping or level of virulence or geographic origin. The dendrogram generated from these isolates based on PCR analysis with five RAPD-PCR primers showed high levels of genetic similarity among the isolates from the same geographical locations. There was partially relationship between the genetic similarity and AGs or level of virulence or geographic origin based on RAPD dendrogram. These results demonstrate that RAPD technique is a useful tool in determining the genetic characterization among isolates of R. solani.

Nitrification of ammonium-rich sanitary landfill leachate.

The nitrification of ammonium-rich wastewater is considered challenging due to the substrate inhibition particularly in the form of free ammonia (FA) and free nitrous acid (FNA) in ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). The feasibility of the nitrifying activated sludge system to completely nitrify synthetic stabilized landfill leachate with N-NH(4)(+) concentration of 1452mg/L was tested in this study. The process started with 0.4kg N-NH(4)(+)/m(3)/day of nitrogen loading rate (NLR) in a fed-batch mode to avoid any accumulation of the FA and FNA in the system followed by increasing the nitrogen loading rate (NLR) gradually. Complete nitrification was achieved with a very high ammonium removal percentage (approximately 100%). The maximum specific and volumetric nitrification rate obtained were 0.49g N-NH(4)(+)/g VSS/day and 3.0kg N-NH(4)(+)/m(3)/day, respectively which were higher than those reported previously for ammonium-rich removal using activated sludge system. The nitrifying sludge exhibited good settling characteristics of up to 36mL/g VSS and a long SRT of more than 53 days which contributed to the success of the nitrification process. The coexistence and syntrophic association of the AOB and NOB was observed by using Fluorescence in situ hybridization (FISH) technique which supported the results on complete nitrification obtained in the system. These findings would be of prominent importance for further treatment of actual sanitary landfill leachate.

Measuring organic carbon, nutrients and heavy metals in rivers receiving leachate from controlled and uncontrolled municipal solid waste (MSW) landfills.

Since landfilling is the common method of waste disposal in Malaysia, river water is greatly exposed to the risk of contamination from leachate unless proper leachate management is carried out. In this study, leachates from three different types of landfills, namely active uncontrolled, active controlled and closed controlled, were characterized, and their relationships with river water chemistry were examined monthly for a year. The influence of leachate on river water chemistry from each type of landfill depended on many factors, including the presence of a leachate control mechanism, leachate characteristics, precipitation, surface runoff and the applied treatment. The impact of leachate from an active uncontrolled landfill was the highest, as the organic content, NH(4)(+)-N, Cd and Mn levels appeared high in the river. At the same time, influences of leachate were also observed from both types of controlled landfills in the form of inorganic nitrogen (NH(4)(+)-N, NO(3)(-)-N and NO(2)(-)-N) and heavy metals (Fe, Cr, Ni and Mn). Improper treatment practice led to high levels of some contaminants in the stream near the closed controlled landfill. Meanwhile, the active controlled landfill, which was located near the coastline, was exposed to the risk of contamination resulting from the pyrite oxidation of the surrounding area.