Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås, Norway.
VEAS WWTP, Slemmestad, Norway.
mBio. 2022 Jun 28;13(3):e0078822. doi: 10.1128/mbio.00788-22. Epub 2022 May 31.
Manipulating soil metabolism through heavy inoculation with microbes is feasible if organic wastes can be utilized as the substrate for growth and vector as a fertilizer. This, however, requires organisms active in both digestate and soil (generalists). Here, we present a dual enrichment strategy to enrich and isolate such generalists among NO-respiring bacteria (NRB) in soil and digestates, to be used as an inoculum for strengthening the NO-reduction capacity of soils. The enrichment strategy utilizes sequential batch enrichment cultures alternating between sterilized digestate and soil as substrates, with each batch initiated with limited O and unlimited NO. The cultures were monitored for gas kinetics and community composition. As predicted by a Lotka-Volterra competition model, cluster analysis identified generalist operational taxonomic units (OTUs) which became dominant, digestate/soil-specialists which did not, and a majority that were gradually diluted out. We isolated several NRBs circumscribed by generalist OTUs. Their denitrification genes and phenotypes predicted a variable capacity to act as NO-sinks, while all genomes predicted broad catabolic capacity. The latter contrasts with previous attempts to enrich NRB by anaerobic incubation of unsterilized digestate only, which selected for organisms with a catabolic capacity limited to fermentation products. The two isolates with the most promising characteristics as NO sinks were Pseudomonas sp. with a full-fledged denitrification-pathway and a sp. carrying only NO reductase (clade II), and soil experiments confirmed their capacity to reduce NO-emissions from soil. The successful enrichment of NRB with broad catabolic spectra suggests that the concept of dual enrichment should also be applicable for enrichment of generalists with traits other than NO reduction. NO emissions from farmed soils are a major source of climate forcing. Here, denitrifying bacteria act as both source and sink for NO, determined by regulatory traits or the absence of genes coding for the enzymes producing or reducing NO. One approach to reducing emissions is to amend large numbers of NO-reducing bacteria (NRB) to soil. This was shown to be feasible by growing NRB to high densities in organic wastes and then applying them as fertilizers. The effect on NO emissions, however, was transient because the isolated NRBs were unsuited to soil. Here, we have developed an enrichment strategy selecting for organisms with generalist lifestyles, tolerant of rapid environmental changes. This was used to isolate robust NRBs that grow both in digestate and when amended to soils. This strategy opens an avenue for obtaining not just robust NRBs to reduce NO emissions, but any organism destined for application to complex environments.
如果可以利用有机废物作为生长的基质并将其作为肥料载体，通过大量接种微生物来操纵土壤代谢是可行的。然而，这需要在消化物和土壤中都具有活性的生物体（广义类群）。在这里，我们提出了一种双重富集策略，即在土壤和消化物中的非呼吸硝酸盐细菌（NRB）中富集和分离这种广义类群，用作强化土壤硝酸盐还原能力的接种物。该富集策略利用连续批量富集培养，以灭菌的消化物和土壤作为交替的基质，每批培养物均以有限的 O 和无限的 NO 开始。培养物的气体动力学和群落组成进行了监测。正如 Lotka-Volterra 竞争模型所预测的那样，聚类分析确定了占主导地位的广义类群操作分类单元（OTU）、不占主导地位的消化物/土壤专业菌以及逐渐被稀释的大多数菌。我们分离出了几个由广义类群 OTU 界定的 NRB。它们的反硝化基因和表型预测了作为硝酸盐汇的可变能力，而所有基因组都预测了广泛的代谢能力。这与以前仅通过厌氧孵育未灭菌消化物来富集 NRB 的尝试形成了鲜明对比，后者仅选择具有代谢能力仅限于发酵产物的生物体。作为硝酸盐汇最有前景的两个特征的分离物是具有完整反硝化途径的 Pseudomonas sp. 和仅携带硝酸盐还原酶（II 类）的 sp.，土壤实验证实了它们减少土壤中硝酸盐排放的能力。广泛代谢谱的 NRB 的成功富集表明，双重富集的概念也应该适用于富集具有除硝酸盐还原以外的其他特性的广义类群。农田土壤中的硝酸盐排放是气候强迫的主要来源。在这里，反硝化细菌既是硝酸盐的源又是硝酸盐的汇，这取决于调节特性或缺乏编码产生或减少硝酸盐的酶的基因。减少排放的一种方法是向土壤中添加大量的硝酸盐还原细菌（NRB）。通过在有机废物中大量培养 NRB 并将其用作肥料来证明这是可行的。然而，这种方法对硝酸盐排放的影响是暂时的，因为分离的 NRB 不适合土壤。在这里，我们开发了一种富集策略，用于选择具有广谱生活方式的生物体，这些生物体能够耐受快速的环境变化。这用于分离既可以在消化物中生长，又可以在添加到土壤中时生长的稳健 NRB。该策略为获得不仅可以减少硝酸盐排放的稳健 NRB，而且还可以获得任何注定要应用于复杂环境的生物体开辟了一条途径。
