Emilie Courtens

Optimized Cryopreservation of Mixed Microbial Communities for Conserved Functionality and Diversity

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Control of nitratation in an oxygen-limited autotrophic nitrification/denitrification rotating biological contactor through disc immersion level variation

With oxygen supply playing a crucial role in an oxygen-limited autotrophic nitrification/denitrification (OLAND) rotating biological contactor (RBC), its controlling factors were investigated in this study. Disc rotation speeds (1.8 and 3.6rpm) showed no influence on the process performance of a lab-scale RBC, although abiotic experiments showed a significant effect on the oxygenation capacity. Estimations of the biological oxygen uptake rate revealed that 85-89% of the oxygen was absorbed by the microorganisms during the air exposure of the discs. Indeed, increasing the disc immersion (50 to 75-80%) could significantly suppress undesired nitratation, on the short and long term. The presented results demonstrated that nitratation could be controlled by the immersion level and revealed that oxygen control in an OLAND RBC should be predominantly based on the atmospheric exposure percentage of the discs.

Efficient total nitrogen removal in an ammonia gas biofilter through high-rate OLAND.

Ammonia gas is conventionally treated in nitrifying biofilters; however, addition of organic carbon to perform post-denitrification is required to obtain total nitrogen removal. Oxygen-limited autotrophic nitrification/denitrification (OLAND), applied in full-scale for wastewater treatment, can offer a cost-effective alternative for gas treatment. In this study, the OLAND application thus was broadened toward ammonia loaded gaseous streams. A down flow, oxygen-saturated biofilter (height of 1.5 m; diameter of 0.11 m) was fed with an ammonia gas stream (248 ± 10 ppmv) at a loading rate of 0.86 ± 0.04 kg N m(-3) biofilter d(-1) and an empty bed residence time of 14 s. After 45 days of operation a stable nitrogen removal rate of 0.67 ± 0.06 kg N m(-3) biofilter d(-1), an ammonia removal efficiency of 99%, a removal of 75-80% of the total nitrogen, and negligible NO/N(2)O productions were obtained at water flow rates of 1.3 ± 0.4 m(3) m(-2) biofilter section d(-1). Profile measurements revealed that 91% of the total nitrogen activity was taking place in the top 36% of the filter. This study demonstrated for the first time highly effective and sustainable autotrophic ammonia removal in a gas biofilter and therefore shows the appealing potential of the OLAND process to treat ammonia containing gaseous streams.

A robust nitrifying community in a bioreactor at 50 °C opens up the path for thermophilic nitrogen removal.

The increasing production of nitrogen-containing fertilizers is crucial to meet the global food demand, yet high losses of reactive nitrogen associated with the food production/consumption chain progressively deteriorate the natural environment. Currently, mesophilic nitrogen-removing microbes eliminate nitrogen from wastewaters. Although thermophilic nitrifiers have been separately enriched from natural environments, no bioreactors are described that couple these processes for the treatment of nitrogen in hot wastewaters. Samples from composting facilities were used as inoculum for the batch-wise enrichment of thermophilic nitrifiers (350 days). Subsequently, the enrichments were transferred to a bioreactor to obtain a stable, high-rate nitrifying process (560 days). The community contained up to 17% ammonia-oxidizing archaea (AOAs) closely related to ‘Candidatus Nitrososphaera gargensis’, and 25% nitrite-oxidizing bacteria (NOBs) related to Nitrospira calida. Incorporation of 13C-derived bicarbonate into the respective characteristic membrane lipids during nitrification supported their activity as autotrophs. Specific activities up to 198±10 and 894±81 mg N g−1 VSS per day for AOAs and NOBs were measured, where NOBs were 33% more sensitive to free ammonia. The NOBs were extremely sensitive to free nitrous acid, whereas the AOAs could only be inhibited by high nitrite concentrations, independent of the free nitrous acid concentration. The observed difference in product/substrate inhibition could facilitate the development of NOB inhibition strategies to achieve more cost-effective processes such as deammonification. This study describes the enrichment of autotrophic thermophilic nitrifiers from a nutrient-rich environment and the successful operation of a thermophilic nitrifying bioreactor for the first time, facilitating opportunities for thermophilic nitrogen removal biotechnology.

Nitric oxide preferentially inhibits nitrite oxidizing communities with high affinity for nitrite

The prerequisite to the development success of the novel mainstream processes partial nitritation/anammox is the out-selection of nitrite oxidizing bacteria (NOB). A recent study suggested that this could be achieved through NO production by ammonium oxidizing bacteria under cyclic oxic-anoxic conditions. Indeed, it is known that among NOB, Nitrobacter species are reversibly inhibited by NO. However, the effect of NO on the activity of the NOB genus Nitrospira is not studied so far. Such an understanding is needed, since Nitrospira related NOB are mostly prevailing in sewage treatment plants. This study quantified the effect of NO on the nitratation activity of sludge types with different Nitrobacter/Nitrospira ratios. In an oxic bubbling column, a dosage of 4.4 mg NO L(-1) d(-1) (∼2 μg NO-N L(-1) in liquid phase) inhibited the Nitrobacter dominated sludge with 24%. For the Nitrospira dominated sludge types, the inhibition was strongly correlated with the nitrite half saturation constant (K(s)) ranging from 0% to 30-50% and 60-80% inhibition of the nitrite oxidation for K(s) of 0.72, 0.36 and 0.06 mg NO2(-)-N L(-1), respectively. This study showed that nitrifying communities with high affinity for nitrite and low specific nitrite oxidation rates (K-strategists) can be strongly inhibited by NO. The degree of inhibition could be confirmed in a set-up with NO dosage through an artificial alginate-based biofilm, ensuring a more direct contact between NO and the microorganisms.

When the smoke disappears: dealing with extinguishing chemicals in firefighting wastewater

Water is not enough. Nowadays, numerous chemicals are used for fire extinction. After use, however, these may unintentionally enter sewerage systems. In order to safely treat firefighting wastewater (FFWW), knowledge of the potential effects of these chemicals on biological treatment processes is essential. This study characterized and mimicked the composition of FFWW containing two powders, three foams and one foam degrader. Nitrogen (162-370 mg NH4(+)-N L(-1)) and phosphorus (173-320 mg PO4(3-)-P L(-1)) concentrations exceeded discharge limits, whereas chemical and biological oxygen demand, suspended solids and detergent concentrations remained sufficiently low. Adequate nutrient removal could be obtained through FeCl3 addition and nitrification/denitrification with acetate as substrate. In batch tests, residual nitrifying activities of 84, 81, 89, 95 and 93% were observed in the presence of powders, foams, foam degrader, synthetic and real FFWW, respectively. All categories showed higher denitrification rates than the control. Although the powders at first seemed to inhibit anammox activity at 82%, after pH correction anammox was fully feasible, allowing nitrogen removal through oxygen-limited nitrification/denitrification (OLAND). Detailed cost calculations indicated that OLAND could save 11% of capital and 68% of operational costs compared to nitrification/denitrification, identifying OLAND as the most recommendable process for nitrogen removal from firefighting wastewaters.