Using FlowCam and molecular techniques to assess the diversity of Cyanobacteria species in water used for food production

Abstract

Globally, the occurrence of cyanobacteria in water currently remains an important subject as they produce cyanotoxins that pose threat to human health. Studies on the contamination of maize meals during mill grinding processes using cyanobacteria-contaminated water have not been conducted. The present study aimed to assess the diversity of cyanobacteria in the samples (process water, uncooked maize meal, and cooked maize meal (porridge)). Polymerized Chain Reaction (PCR) and Advanced digital flow cytometry (FlowCAM) were used to detect and identify cyanobacterial species available in these samples. 16S Primers (forward and reverse) tailed with Universal Sequences were used for amplification and sequencing of full-length 16S rRNA genes from cyanobacteria found in all samples. Cyanobacterial species from order Nostocales, Pseudanabaenales, Oscillatoriales Chroococcales, Synechococcales, and unclassified cyanobacterial order, some of which have the potential to produce cyanotoxins were amplified and identified in process water, raw maize meal and porridge samples using PCR. Images of the genus Microcystis, Phormidium, and Leptolyngbya were captured in process water samples using FlowCAM. These findings show the presence of cyanobacteria species in process water used for maize meal and the absence in cooked maize meal. The presence of cyanobacteria in process water is likely another route of human exposure to cyanotoxins.

Figure 1

Map of the study area.

Figure 2

Agarose gel electrophoresis showing amplified 16S rRNA of the bacterial isolates. Lane ladder represents the 100 bp molecular ladder. The lanes (GFF1, GFF2, P1, P2 and Porridge) express the level of migration of genes on the agarose gel.

Figure 3

Representative FlowCAM images of genus Microcystis sp. (A) obtained from the SD-2 water sample; Leptolyngbya sp. (B) and Phormidium sp. (C) obtained from the SD-1 water sample; and Microcystis sp. (D1-2) obtained from the SM-1 and SM-2 water samples, respectively. All images are < 20 µm in diameter.

Figure 4

Pie charts showing the relative proportion of phyla: (A) (GFF1), (B) (GFF2), (C) (P1), (D) (P2), and (E) (P3); and order: (F) (GFF1) and G (GFF2) amplified by the primers set used in the present study.

Figure 5

Topology of the 16S rRNA gene sequences of the cyanobacterial strains isolated from the present study and their closely related sequences from the NCBI database constructed using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Five base substitutions for nucleotide positions are represented by the scale bar. The sequences obtained in the present study are indicated by red rhombus.