<p>To make bacterial-based self-healing concrete, alkaline-resistant bacterial spores, nutrient sources, and a calcium source are incorporated into a concrete matrix. Two ureolytic spore-forming bacteria, <i>Sporosarcina pasteurii</i>, <i>Lysinibacillus sphaericus</i>, and two non-ureolytic spore-forming bacteria, <i>Bacillus cohnii</i>, and <i>Bacillus pseudofirmus</i>, which have been used in previous studies as bacterial concrete healing agents, were compared in this study. The four bacteria were compared for their (1) sporulation rates on different sporulation agar plates, (2) growth in five liquid media, (3) survival rates in light weight aggregates (LWA) and in mortar samples, and (4) calcium carbonate precipitation rates from either calcium lactate or calcium nitrate. Sporulation was successfully induced after three-day incubation at 30°C on an appropriate sporulation medium. High sporulation rates of <i>B. cohnii</i>, and <i>B. pseudofirmus</i>(93% and 99% respectively) were found on alkaline R2A medium (AR2A). A sporulation rate (89%) of <i>S. pasteruii</i>was observed on tryptic soy agar supplemented with 2% urea (TSAU)<i>.</i>The highest sporulation rate (60%) of <i>L. sphaericus</i>was found on R2A medium supplemented with 2% urea (R2AU). In the growth study, tryptic soy broth supplemented with 2% urea (TSBU) was a positive control which supported rapid growth of all four bacteria. <i>Sporosarcina pasteurii </i>and <i>L. pasteurii</i>showed rapid growth rates in alkaline yeast extract broth (AYE) and yeast extract with 2% urea broth (YEU) respectively. In contrast, <i>B. cohnii</i>, and <i>B. pseudofirmus</i>grew poorly in all media except in the positive control. Viable counts of the four bacterial spores reduced (1.8–3.3 logs) during the first 24 h in mortar samples and then remained stable for next 27 days testing period. Among the four, <i>S. pasteurii</i>showed the smallest reduction of viable counts (1.8–2.5 logs) in mortar after one day of incubation. Both <i>S. pasteurii</i>and <i>L. sphaericus</i>showed high CaCO₃productions (>80%) after 24 h incubation at 30°C in YEU containing either calcium nitrate or calcium lactate. However, <i>B. pseudofirmus</i>and <i>B. cohnii </i>showed<i></i>low calcite recovery rates (<11%) in AYE containing either<i></i>calcium nitrate or calcium lactate under the same incubation condition. Overall, <i>S. pasteurii</i>was the best bacterial concrete healing agent of the four. This bacterium had (1) rapid growth rate in AYE, (2) about 90% sporulation rate within 3 days, (3) highest survival rates after 24 h in mortar samples and, (4) high CaCO₃precipitation rates, 82 or 98%, in broth containing calcium nitrate or calcium lactate respectively.</p><p>In addition, two different lightweight aggregates (LWA), expanded shale (ES) and expanded clay (EC), which were used as bacterial carriers and protective materials, were compared in this study. Each type of LWA was separated into three sizes (<0.85 mm, 0.85– 2.0 mm, and >2.0 mm) and immobilized with spores of <i>B. cohnii</i>or <i>B. pseudofirmus.</i>Viable counts recovered from EC and ES reduced <1.0 log after the immobilization process and remained stable during the 150 days testing period. Neither the type nor the particle sizes of the two LWA significantly affected the survival rates of the bacterial spores. This result showed that both EC and ES could be used as carriers for bacterial healing agents. It was also found that when the spores were immobilized with nutrients in LWA, their survival rates in mortar samples can be improved slightly (<1.0 log).</p><p><br></p>