This study has contributed to the patented technology of biocement (Microbial Biocementation, WO/2006/066326). Biocementation or biogrout is a sand consolidation technology, in which the carbonate released from microbial urea hydrolysis precipitates with an excess of calcium ions to form in-situ calcite (CaCO3) precipitation. Under the right conditions this can result in soil solidification and has found significant commercial interest.ud udThis study has enriched and isolated highly urease active bacteria, particularly suitable for the fermentation process. Six strains with different properties relevant for biocementation were isolated. The most urease active strain (strain MCP11) produced sufficient urease to allow the use of the non-concentrated cell suspension for biocementation experiments. Activities and specific activities were 11-28 mM urea hydrolysed.min-1 and 2.2-5.6 mM urea hydrolysed.min-1.OD-1 respectively. A separate strain (strain MCP4) showed spontaneous flocculation at the end of the batch growth, showing its increased tendency to attach to surfaces. This can be useful for effective cell concentration and for improved attachment during the cementation process.ud udThe possibility of causing cementation by using enrichments rather than pure strains has been documented. This may allow a cheaper production of the urease than by traditional pure culture processes.ud udUrease production was optimised by increasing the concentration of yeast extract and the addition of Ni2+ ions to the growth media, resulting in increasing urease activity as the reproducible urease yield. This was accomplished by the addition of 10 μM Ni 2+ ions and increasing the level of yeast extract to 20 g.L-1ud udSome of the isolated strains were suitable for biocementation process producing mechanical strength (≥ 0.6 MPa) within several hours depending on the rate of urea conversion. This mechanical strength enhancement of the cemented columns was performed without a large decrease in the permeability.ud udThe formation of CaCO3 crystals in the presence of high concentration of calcium and urea was monitored. This crystal growth was monitored over time by video recording the ureolytic reaction on a microscopic slide. The crystals also were examined through SEM. It was found that two types of CaCO3 precipitates were formed; these precipitates were calcite rhombohedral crystals and spheroids. Video clips showed that the rhombohedral crystals originated from the spheroids. These spheroids were fragile, not stable and were considered to be vaterite.ud udThis study suggested that the strength of the cemented column was caused mostly due to the point-to-point contacts of rhombohedral CaCO3 crystals and adjacent sand grains.ud udA method of producing high strength cemented samples from sand was developed. This method first attaches the cells into the sand-column by growing them in the presence of calcium ions as little as 6 mM. Then, the cells were incubated in-situ for about 48 hours to enable attachment to the surface of the sand granules. Then the cells were reused over 20-times by continuous supply of cementation solution (equi-molar concentration of calcium and urea). This method produced a mechanical strength of up to 30 MPa, which is equivalent to construction cement.ud udThe mechanical strength could be increased by supplying the bacteria in-situ with a food source and 10 μM Ni2+ ions, allowing some measures of reaction rate control in-situ. To our knowledge, this study was the first study to use biological cementation to produce strength comparable to that of traditional cemented construction materials such as sandstone and concrete.ud udThe key factors for the optimal CaCO3 precipitation (strength production) in-situ were examined. It was found that in-situ urease activity was the key factor for strength production. The maximum in-situ urease activity was achieved by supplementing the cementation solution with growth media, and the use of 0.5 M urea and Ca2+ as cementation solution. The in-situ urease activity differed according to the different bacterial strains which tolerated the cementation conditions differently.ud udOne of the advantages of the present study was that cementation of porous media could be achieved without clogging the injection end. The injection end could be clogged by CaCO3 precipitation due to cementation reaction (cells, calcium and urea). By sequentially flushing the cells and cementation solution, clogging of the injection end could be avoided and high penetration depth was achieved as long as there was sufficient passage of cementation solution. Uniform cementation along 1 m packed sand-column was obtained. This uniformity was confirmed by the urease activity measurement, calcite precipitation and mechanical strength production. For finer sand, homogenous cementation proved more difficult.
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机译：这项研究为生物水泥的专利技术做出了贡献（微生物生物水泥，WO / 2006/066326）。生物胶结或生物灌浆是一种固砂技术，其中微生物尿素水解释放的碳酸盐与过量的钙离子一起沉淀，形成原位方解石（CaCO3）沉淀。在适当的条件下，这可能会导致土壤固化，并且已经找到了重要的商业价值。分离了具有与生物胶凝有关的不同性质的六个菌株。最具脲酶活性的菌株（MCP11菌株）产生了足够的脲酶，可以将未浓缩的细胞悬液用于生物固结实验。活性和比活分别为11-28mM尿素水解.min-1和2.2-5.6mM尿素水解.min-1.OD-1。单独的菌株（MCP4菌株）在分批生长结束时显示出自发絮凝，表明其附着在表面上的趋势增加。这对于有效的细胞浓度和在胶结过程中改善附着力可能是有用的。 ud ud已记录了通过使用富集而不是纯菌株引起胶结的可能性。与传统的纯培养方法相比，这可能使尿素酶的生产更便宜。通过增加酵母提取物的浓度和向生长培养基中添加Ni2 +离子，可以优化尿素酶的生产，从而增加了尿素酶作为可再生尿素酶的活性。让。这是通过添加10μMNi 2+离子并将酵母提取物的水平提高到20 gL-1 ud ud来完成的。有些分离的菌株适合于数小时内产生机械强度（≥0.6 MPa）的生物胶凝过程，具体取决于尿素转化率在不大幅降低渗透率的情况下进行了水泥柱的这种机械强度增强。 ud ud在高浓度钙和尿素存在下，监测CaCO3晶体的形成。通过在显微镜载玻片上记录尿素分解反应，随时间监测晶体的生长。还通过SEM检查晶体。发现形成了两种类型的CaCO 3沉淀物；即CaCO 3沉淀物。这些沉淀物是方解石菱面体晶体和球体。视频剪辑显示，菱面体晶体起源于球体。这些球状体易碎，不稳定，被认为是球ate石。开发了一种用砂子生产高强度胶结样品的方法。该方法首先通过在低至6 mM的钙离子存在下使其生长，将细胞附着在沙柱上。然后，将细胞在原位温育约48小时，以使其附着在沙粒表面。然后通过连续供应胶结溶液（钙和尿素的摩尔浓度）将细胞重复使用20次以上。这种方法产生的机械强度高达30 MPa，相当于建筑水泥。 ud ud可以通过向细菌原位提供食物源和10μMNi2 +离子来增加机械强度，从而可以采取一些反应措施。速率控制。据我们所知，该研究是第一个使用生物胶结来产生与传统胶结建筑材料（如砂岩和混凝土）相当的强度的研究。 ud ud最佳就地沉淀CaCO3（强度产生）的关键因素是检查。发现原位脲酶活性是产生强度的关键因素。通过向胶结溶液中添加生长介质，并使用0.5 M尿素和Ca2 +作为胶结溶液，可以实现最大的原位脲酶活性。根据不同的细菌菌株，原位脲酶活性会有所不同，对胶结条件的耐受程度也不同。 ud ud本研究的优势之一是可以实现多孔介质的胶结而不堵塞注射端。由于胶结反应（细胞，钙和尿素），CaCO3沉淀可能会堵塞注入端。通过依次冲洗细胞和胶结溶液，可以避免注入端的堵塞，并且只要胶结溶液有足够的通道，就可以实现高渗透深度。获得了沿1 m堆积砂柱的均匀胶结作用。通过尿素酶活性测量，方解石沉淀和机械强度产生来确认这种均匀性。对于更细的砂子，均匀的胶结更加困难。
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