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Microalgae are a sustainable bioresource, and the biofuel they produce is widely considered to be an alternative to limited natural fuel resources. However, microalgae harvesting is a bottleneck in the development of technology. Axenic Chlorella vulgaris microalgae exhibit poor harvesting, as expressed by a flocculation efficiency of 0·2%. This work optimized the co-culture conditions of C. vulgaris and bioflocculant-producing bacteria in synthetic wastewater using response surface methodology (RSM), thus aiming to enhance C. vulgaris harvesting and lipid content. Three significant process variables— inoculation ratio of bacteria and microalgae, initial glucose concentration, and co-culture time— were proposed in the RSM model. F-values (3·98/8·46) and R2 values (0·7817/0·8711) both indicated a reasonable prediction by the RSM model. The results showed that C. vulgaris harvesting efficiency reached 45·0–50·0%, and the lipid content was over 21·0% when co-cultured with bioflocculant-producing bacteria under the optimized culture conditions of inoculation ratio of bacteria and microalgae of 0·20–0·25, initial glucose concentration of <1·5 kg m−3 and co-culture time of 9–14 days. This work provided new insights into microalgae harvesting and cost-effective microalgal bioproducts, and confirmed the promising prospect of introducing bioflocculant-producing bacteria into microalgae bioenergy production.