Hyaluronan (HA) synthesis by microalgal Chlorella cells in combination with chloroviruses represents a unique eco-friendly process for converting solar energy and CO2 into useful materials. However, at the final stage of viral infection, infected host cells are completely lysed, and thus HA should be harvested before cell lysis. In the current study, two methods were investigated to improve the yield of HA: (i) adopting slow-growing chlorovirus isolates and (ii) modification of the virus replication process using an inhibitor of DNA synthesis, aphidicolin. Compared with Paramecium bursaria Chlorella virus type 1 (PBCV-1), the prototype chlorovirus, slow-growing virus isolates (CVO1 and CVTS1) produced a 1.5 times higher concentration of HA in infected Chlorella cultures. Furthermore, addition of aphidicolin, an inhibitor of DNA synthesis, delayed virus replication and increased the final concentration of HA 1.5-fold that of cultures without the addition of aphidicolin. Therefore, a 2- to 3-fold increase in the yield of HA by the Chlorella-virus system was attained by using slow-growing viral isolates and the addition of aphidicolin. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.