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It is generally accepted that the intestinal microbiota plays an important role in sustaining health and productivity of animals. Chlorella vulgaris, a naturally occurring green microalga, is believed to influence performance and health, including bird reproduction and egg quality. The nutritive value of open or indoor cultured C. vulgaris depends upon the technological process used to treat the algal mass. In the present paper, it is presented and discussed how 2 differentially processed C. vulgaris powders (spray-dried: SD-CV; bullet-milled and spray-dried: BMSD-CV) affected crop and cecal microbiota in laying hens. Polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA gene fragments was applied. The diversity of the crop universal bacterial DGGE fingerprints was not affected (6.4 +/- 1.65, 5.4 +/- 1.19, and 5.5 +/- 1.35 in the control, SD-CV, and BMSD-CV, respectively). Most of the bands from the corresponding positions in the gels were closely related to Lactobacillus sp. The DGGE fingerprints of V2-V3 fragments of 16S rRNA of crop lactobacilli had lower diversity in the control hens (8.7 +/- 1.22) than in the SD-CV (9.2 +/- 1.77) and BMSD-CV (9.9 +/- 1.88); thus, feeding C. vulgaris resulted in increased lactobacilli diversity in crop. A band closely related to Lactobacillus ingluviei was present in 9 out of 12 hens in the control group but in only 1 bird in the SD-CV and in 5 out of 11 birds in the BMSD-CV, suggesting a negative effect of C. vulgaris on this lactobacillus. Feeding C. vulgaris to laying hens also resulted in increased bacterial community diversity in the ceca. No effect of the technological processing of the microalgae on the microbial diversity could be observed. The diversity of the ceca universal bacterial DGGE fingerprints was lower in the control group than in the SD-CV and BMSD-CV (5.6 +/- 1.72 vs. 9.16 +/- 2.64 and 9.31 +/- 2.41, respectively). Most of the sequences retrieved from the DGGE bands formed ceca that were closely related to Ruminococcaceae, Lachnospiraceae, and lactobacilli, giving further insight into still poorly discovered intestinal microbiota of laying hens.