Microbial CaCO3 Precipitation: For the Production of Biocement
Microbial CaCO3 Precipitation: For the Production of Biocement
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2008-06
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通讯作者：
Victoria S. Whiffin
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作者：
Victoria S. Whiffin
广泛分布的脲酶对尿素的水解是特殊的，因为它是为数不多的可以产生碳酸盐离子而不产生质子的生物反应之一。当这种水解在富钙环境中发生时，方解石（碳酸钙）从溶液中析出，形成固态结晶材料。沉淀晶体的结合强度高度依赖于碳酸盐形成的速率，在适当的条件下，可以控制反应生成硬结合方解石水泥（或生物水泥）。本论文的目的是开发一种工业上适合的具有成本效益的微生物工艺来生产脲酶活性细胞，并研究脲酶活性细胞作为生物水泥生产催化剂的潜力。比较了两种适宜菌株的生物胶结能力。与Proteus vulgaris相比，Sporosarcina pasteurii（正式的巴氏芽孢杆菌）产生的脲酶活性水平明显更高，但脲酶活性水平随生物量的变化而变化，这表明该酶不是文献所述的组成型酶，而是受调节的。研究了巴氏杆菌最大脲酶活性的环境和生理条件，发现该菌的潜在脲酶容量非常高（29 mM脲- min-1）。OD-1)，足以进行生物胶结，无需额外处理（如浓缩、细胞裂解）。本研究并未完全阐明巴氏杆菌脲酶的调控机制，但表明脲酶特异性活性低并非由于尿素耗竭或主要反应产物铵浓度高所致。pH条件对脲酶有调节作用，但显然存在另一种协同调节机制。尽管没有充分利用巴氏杆菌的脲酶能力，但仍然可以获得足够的脲酶活性，允许直接应用酶而无需额外处理，并且认为该生物适合生物胶结。脲酶是胶结过程中最昂贵的组分，并且需要具有成本效益的生产，因此开发了一种用于大规模培养巴氏杆菌的经济增长程序。该菌是一种中等亲碱菌（生长最适pH为9.25），在非无菌条件下，只需最少的上游和下游加工，就能培养出足够的生物胶结活性。培养基是经济的，昂贵的成分被食品级蛋白质来源和醋酸盐取代，这降低了95%的生产成本。高水平的脲酶活性(21毫米尿素水解。min-1)在新介质中以较低的成本（每升0.20美元）生产。在生物胶结条件下（即高浓度尿素、Ca2+、NH4 +/NH3、NO3 -和Cl-离子的存在），对整个巴氏杆菌细胞中脲酶的性能进行了评估。结果表明，在胶结过程中，尿素的水解速率不是恒定的，而是在很大程度上受外部尿素和钙浓度的控制，在胶结过程中，由于固体碳酸钙从体系中析出，尿素和钙的浓度不断变化。生成了一个简单的模型来预测在胶结过程中尿素水解率的变化。结果表明，整个细胞的巴氏杆菌脲酶耐受浓度高达3 M的尿素和2 M的钙，且尿素水解速率不受3 M铵的影响。这允许在一次处理中控制高达1.5 M CaCO3的沉淀，并表明酶在极端化学条件下非常稳定。开发了一种经济高效的高胶结强度固井工艺。通过确定尿素水解率对强度发展的影响，进行了几次生物胶结试验，以优化所赋予的胶结强度。结果表明，在较低的尿素水解速率下产生较高的胶结强度，胶结强度的发展在整个反应过程中不是线性的，而主要发生在反应的前几个小时。此外，整个细胞细菌酶具有固定在胶结材料中并在后续应用中重复使用的能力，从而大大节省了该过程的成本。进行了一项行业赞助的试验，以调查生物水泥增加两种不同砂质土壤的原位强度和刚度的有效性；(a) Koolschijn砂和(b) 90% Koolschijn砂与10%泥炭混合（荷兰文）。生物胶结处理后，Koolschijn砂抗剪强度为1.8 MPa，刚度为250 MPa，强度分别比未固结砂提高8倍和3倍。在与泥炭混合的沙子中观察到明显较低的强度改善。综合而言，在经济上可接受的条件下生产细菌的试验和胶结试验支持了现场生产和大型油田现场应用的可能性。
The hydrolysis of urea by the widely distributed enzyme urease is special in that it is one of the few biologically occurring reactions that can generate carbonate ions without an associated production of protons. When this hydrolysis occurs in a calcium-rich environment, calcite (calcium carbonate) precipitates from solution forming a solid-crystalline material. The binding strength of the precipitated crystals is highly dependent on the rate of carbonate formation and under suitable conditions it is possible to control the reaction to generate hard binding calcite cement (or Biocement). The objective of this thesis was to develop an industrially suitable cost-effective microbial process for the production of urease active cells and investigate the potential for urease active cells to act as a catalyst for the production of Biocement. The biocementation capability of two suitable strains was compared. Sporosarcina pasteurii (formally Bacillus pasteurii) produced significantly higher levels of urease activity compared to Proteus vulgaris, however the level of urease activity was variable with respect to biomass suggesting that the enzyme was not constitutive as indicated by the literature, but subject to regulation. The environmental and physiological conditions for maximum urease activity in S. pasteurii were investigated and it was found that the potential urease capacity of the organism was very high (29 mM urea.min-1.OD-1) and sufficient for biocementation without additional processing (e.g. concentration, cell lysis). The regulation mechanism for S. pasteurii urease was not fully elucidated in this study, however it was shown that low specific urease activity was not due to depletion of urea nor due to the high concentrations of the main reaction product, ammonium. pH conditions were shown to have a regulatory effect on urease but it was evident that another co-regulating mechanism existed. Despite not fully exploiting the urease capability of S. pasteurii, sufficient urease activity to allow direct application of the enzyme without additional processing could still be achieved and the organism was considered suitable for biocementation. Urease was the most expensive component of the cementation process and cost-efficient production was desired, thus an economic growth procedure was developed for large-scale cultivation of S. pasteurii. The organism is a moderate alkaliphile (growth optimum pH 9.25) and it was shown that sufficient activity for biocementation could be cultivated in non-sterile conditions with a minimum of upstream and downstream processing. The cultivation medium was economised and expensive components were replace with a food-grade protein source and acetate, which lowered production costs by 95%. A high level of urease activity (21 mM urea hydrolysed.min-1) was produced in the new medium at a low cost ($0.20 (AUD) per L). The performance of urease in whole S. pasteurii cells was evaluated under biocementation conditions (i.e. presence of high concentrations of urea, Ca2+, NH4 +/NH3, NO3 - and Cl- ions). It was established that the rate of urea hydrolysis was not constant during cementation, but largely controlled by the external concentrations of urea and calcium, which constantly changed during cementation due to precipitation of solid calcium carbonate from the system. A simple model was generated that predicted the change in urea hydrolysis rate over the course of cementation. It was shown that whole cell S. pasteurii urease was tolerant to concentrations of up to 3 M urea and 2 M calcium, and the rate of urea hydrolysis was unaffected up to by 3 M ammonium. This allowed the controlled precipitation of up to 1.5 M CaCO3 within one treatment, and indicated that the enzyme was very stable inspite of extreme chemical conditions. A cost-efficient cementation procedure for the production of high cementation strength was developed. Several biocementation trials were conducted into order to optimise the imparted cementation strength by determining the effect of urea hydrolysis rate on the development of strength. It was shown that high cementation strength was produced at low urea hydrolysis rates and that the development of cementation strength was not linear over the course of the reaction but mostly occurred in the first few hours of the reaction. In addition, the whole cell bacterial enzyme had capacity to be immobilised in the cementation material and re-used to subsequent applications, offering a significant cost-saving to the process. An industry-sponsored trial was undertaken to investigate the effectiveness of Biocement for increasing in-situ strength and stiffness of two different sandy soils; (a) Koolschijn sand and (b) 90% Koolschijn sand mixed with 10% peat (Holland Veen). After biocementation treatment, Koolschijn sand indicated a shear strength of 1.8 MPa and a stiffness of 250 MPa, which represents an 8-fold and 3-fold respective improvement in strength compared to unconsolidated sand. Significantly lower strength improvements were observed in sand mixed with peat. In combination, trials of producing bacteria under economically acceptable conditions and cementation trials support the possibility of on-site production and in-situ application of large field applications.