A selection of Avecom's peer-reviewed publications.

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The environmental impact of the dairy industry is heavily influenced by the overproduction of nitrogen- and carbon-rich effluents. The present study proposes an innovative process to recover waste contaminated nitrogen from anaerobic digestate while treating excess cheese whey (CW) and producing high-quality, clean single cell protein (SCP). By relying on direct aeration stripping techniques, employing an airflow subsequently used in the aerobic cheese whey fermentation step, the investigated process was able to strip 41–80% of the total ammonium nitrogen (N-NH4+) from liquid digestate. The stripped ammonia gas (NH3) was completely recovered as N-NH4+ in the acidic CW, and further upcycled into SCP having a total protein content of 74.7% and a balanced amino acids profile. A preliminary techno-economic analysis revealed the potential to directly recover and upcycle nitrogen into SCP at costs (4.3–6.3 €·kgN−1) and energetic inputs (90–132 MJ·kgN−1) matching those of conventional feed and nitrogen management processes.

Carbon and nitrogen present in residual water streams can be converted into microbial protein and used as animal feed in aquaculture. While microbial protein is thought to be more environmentally sustainable when compared to proteins made from fish residues or plants, nothing is known about how it performs in an absolute sustainability perspective, relative to planetary boundaries. Here, a systems-oriented analysis using life cycle assessment (LCA) linked to the planetary boundaries framework was conducted to assess environmental performance of a pilot-scale microbial protein production from starch-rich process water using aerobic heterotrophs. Results showed that while this microbial-protein indeed performed significantly better than just fishmeal or soybean meal for impacts related to nitrogen (N) and phosphorus (P) flows, none of the three feeds were found sustainable in relation to all planetary boundaries. This constitutes an opportunity for technology developers when the microbial protein production is scaled up and matures.

Book Chapter: Chapter 2: Resource recovery from industrial wastewater: what and how much is there?

Resource Recovery from Water: Principles and Application (pp.Chapter 2).  Publisher: IWA Publishing. February 2022

Throughout history, the first and foremost role of urban water management has been the protection of human health and the local aquatic environment. To this end, the practice of (waste-)water treatment has maintained a central focus on the removal of pollutants through dissipative pathways. Approaches like – in the case of wastewater treatment – the activated sludge process, which makes ‘hazardous things’ disappear, have benefitted our society tremendously by safeguarding human and environmental health. While conventional (waste-)water treatment is regarded as one of the greatest engineering achievements of the 20th century, these dissipative approaches will not suffice in the 21st century as we enter the era of the circular economy. A key challenge for the future of urban water management is the need to re-envision the role of water infrastructure, still holding paramount the safeguard of human and environmental health while also becoming a more proactive force for sustainable development through the recovery of resources embedded in urban water. This book aims (i) to explain the basic principles governing resource recovery from water (how much is there, really); (ii) to provide a comprehensive overview and critical assessment of the established and emerging technologies for resource recovery from water; and (iii) to put resource recovery from water in a legal, economic (including the economy of scale of recovered products), social (consumer's point of view), and environmental sustainability framework. This book serves as a powerful teaching tool at the graduate entry master level with an aim to help develop the next generation of engineers and experts and is also highly relevant for seasoned water professionals and practicing engineers.

Engineering microbial technologies for environmental sustainability: choices to make

Microbial Biotechnology. 15. 10.1111/1751-7915.13986. December 2021


Microbial technologies have provided solutions to key challenges in our daily lives for over a century. In the debate about the ongoing climate change and the need for planetary sustainability, microbial ecology and microbial technologies are rarely considered. Nonetheless, they can bring forward vital solutions to decrease and even prevent long-term effects of climate change. The key to the success of microbial technologies is an effective, target-oriented microbiome management. Here, we highlight how microbial technologies can play a key role in both natural, i.e. soils and aquatic ecosystems, and semi-natural or even entirely human-made, engineered ecosystems, e.g. (waste) water treatment and bodily systems. First, we set forward fundamental guidelines for effective soil microbial resource management, especially with respect to nutrient loss and greenhouse gas abatement. Next, we focus on closing the water circle, integrating resource recovery. We also address the essential interaction of the human and animal host with their respective microbiomes. Finally, we set forward some key future potentials, such as microbial protein and the need to overcome microphobia for microbial products and services. Overall, we conclude that by relying on the wisdom of the past, we can tackle the challenges of our current era through microbial technologies.

Effective orthophosphate removal strategies are needed to counteract eutrophication and guarantee water quality. Previously, we established that hydrogen-oxidizing bacteria (HOB) have the ability to remove orthophosphate from artificial surface water. In the present study, we expand the application of the HOB orthophosphate removal strategy (1) to treat artificial surface water with low initial orthophosphate concentrations, (2) to treat real surface water and real wastewater effluent, and (3) to remove orthophosphate continuously. For synthetic surface water, irrespective of the initial concentration of 0.7, 0.5, 0.3, and 0.1 mg PO43−-P/L, ultra-low concentrations (0.0058 ± 0.0028 mg PO43−-P/L) were obtained. When artificial surface water was replaced by real surface water, without added nutrients or other chemicals, it was shown that over 90% orthophosphate could be removed within 30 min of operation in a batch configuration (0.031 ± 0.023 mg PO43−-P/L). In continuous operation, orthophosphate removal from surface water left an average concentration of 0.040 ± 0.036 for 60 days, and the lowest orthophosphate concentration measured was 0.013 mg PO43−/L. Simultaneously, nitrate was continuously removed for 60 days below 0.1 mg/L. The ability to remove orthophosphate even under nitrogen limiting conditions might be related to the ability of HOB to fix nitrogen. This study brings valuable insights into the potential use of HOB biofilms for nutrient remediation and recovery.

There is an urgent need for sustainable protein supply routes with low environmental footprint. Recently, the use of hydrogen oxidizing bacteria (HOB) as a platform for high quality microbial protein (MP) production has regained interest. This study aims to investigate the added value of using conditions such as salt and temperature to steer HOB communities to lower diversities, while maintaining a high protein content and a high quality amino acid profile. Pressure drop and hydrogen consumption were measured for 56 days to evaluate autotrophy of a total of six communities in serum flasks. Of the six communities, four were enriched under saline (0.0, 0.25, 0.5 and 1.0 mol NaCl l⁻¹) and two under thermophilic conditions (65°C). Five communities enriched for HOB were subsequently cultivated in continuously stirred reactors under the same conditions to evaluate their potential as microbial protein producers. The protein percentages ranged from 41 to 80%. The highest protein content was obtained for the thermophilic enrichments. Amino acid profiles were comparable to protein sources commonly used for feed purposes. Members of the genus Achromobacter were found to dominate the saline enrichments while members of the genus Hydrogenibacillus were found to dominate the thermophilic enrichments. Here we show that enriching for HOB while steering the community toward low diversity and maintaining a high quality protein content can be successfully achieved, both in saline and thermophilic conditions. IMPORTANCE Alternative feed and food supply chains are required to decrease water and land use. HOB offer a promising substitute for traditional agricultural practice to produce microbial protein (MP) from residual materials and renewable energy. To safeguard product stability, the composition of the HOB community should be controlled. Defining strategies to maintain the stability of the communities is therefore key for optimization purposes. In this study, we use salt and temperature as independent conditions to stabilize the composition of the HOB communities. Based on the results presented, we conclude that HOB communities can be steered to have low diversity using the presented conditions while producing a desirable protein content with a valuable amino acid profile.

Circular management of carbon and nutrients is at the basis of future environmental sustainability and global food safety. However, direct resource recovery and upcycling from wastes pose safety concerns related to source contamination. By carefully evaluating the potential integrations of existing and emerging bio-technologies, we put forward new possible pathways for the clean-tech upcycling of recovered carbon and nutrients. By coupling anaerobic digestion and thermochemical gasification, the key process line converts biowastes to clean gaseous substrates (e.g. H2, CH4, CO2, CO, NH3, P2), useable for the fermentative production of safe single cell protein (SCP). The subsequent direct (aerobic) or two-stage (anaerobic/aerobic) SCP fermentation of energy-rich gases can produce protein-rich microbial biomass. Worldwide, this is estimated to generate up to 606 Mt SCP per year, with the protein content more than 3 times the worldwide annual soybean meal protein production. The combined SCP and biochar production can further capture and store up to 2.33 Gt CO2-eq per year, representing ∼50% of the Paris Agreement target on annual carbon capture. Finally, this approach could recycle up to 18.5 Mt nitrogen per year (∼8% of current N losses) and 6.5 Mt phosphorus per year (∼25% of the annual phosphorus fertilizer production). In view of a low-carbon and sustainable circular bioeconomy, this concept presents high impact and positive implications, especially in terms of safe future feed and food supply. Producing SCP as a multi-applicable recovery product can be already carried out at production costs that are competitive with those of other feed additives, especially if the social cost of carbon emissions is considered.

This paper proposes the use of hydrogen oxidizing bacteria (HOB) for the removal of orthophosphate from surface water as treatment step to prevent cyanobacterial blooms. To be effective as an orthophosphate removal strategy, an efficient transfer of hydrogen to the HOB is essential. A trickling filter was selected for this purpose. Using this system, a removal rate of 11.32 ± 0.43 mg PO4−3-P/L.d was achieved. The HOB biomass, developed on the trickling filter, is composed of 1.25% phosphorus on dry matter, which suggests that the orthophosphate removal principle is based on HOB growth. Cyanobacterial growth assays of the untreated and treated water showed that Synechocystis sp was only able to grow in the untreated water. Orthophosphate was removed to average residual values of 0.008 mg/L. In this proof of principle study, it is shown that HOB are able to remove orthophosphate from water to concentrations that prevent cyanobacterial growth.

Anaerobic digesters produce biogas, a mixture of predominantly CH4 and CO2, which is typically incinerated to recover electrical and/or thermal energy. In a context of circular economy, the CH4 and CO2 could be used as chemical feedstock in combination with ammonium from the digestate. Their combination into protein‐rich bacterial, used as animal feed additive, could contribute to the ever growing global demand for nutritive protein sources and improve the overall nitrogen efficiency of the current agro‐ feed/food chain. In this concept, renewable CH4 and H2 can serve as carbon‐neutral energy sources for the production of protein‐rich cellular biomass, while assimilating and upgrading recovered ammonia from the digestate. This study evaluated the potential of producing sustainable high‐quality protein additives in a decentralized way through coupling anaerobic digestion and microbial protein production using methanotrophic and hydrogenotrophic bacteria in an on‐farm bioreactor. We show that a practical case digester handling liquid piggery manure, of which the energy content is supplemented for 30% with co‐substrates, provides sufficient biogas to allow the subsequent microbial protein as feed production for about 37% of the number of pigs from which the manure was derived. Overall, producing microbial protein on the farm from available methane and ammonia liberated by anaerobic digesters treating manure appears economically and technically feasible within the current range of market prices existing for high‐quality protein. The case of producing biomethane for grid injection and upgrading the CO2 with electrolytic hydrogen to microbial protein by means of hydrogen‐oxidizing bacteria was also examined but found less attractive at the current production prices of renewable hydrogen. Our calculations show that this route is only of commercial interest if the protein value equals the value of high‐value protein additives like fishmeal and if the avoided costs for nutrient removal from the digestate are taken into consideration. Anaerobic digesters are the core engine for a biorefinery producing microbial protein. Either the biogas can be used directly for its production, or via biogas upgrading the separate CO2 can be combined with electrolytic hydrogen to produce more protein.

Sustainable development is driving a rapid focus shift in the wastewater and organic waste treatment sectors, from a “removal and disposal” approach towards the recovery and reuse of water, energy and materials (e.g. carbon or nutrients). Purple phototrophic bacteria (PPB) are receiving increasing attention due to their capability of growing photoheterotrophically under anaerobic conditions. Using light as energy source, PPB can simultaneously assimilate carbon and nutrients at high efficiencies (with biomass yields close to unity (1 g CODbiomass·g CODremoved−1)), facilitating the maximum recovery of these resources as different value-added products. The effective use of infrared light enables selective PPB enrichment in non-sterile conditions, without competition with other phototrophs such as microalgae if ultraviolet-visible wavelengths are filtered. This review reunites results systematically gathered from over 177 scientific articles, aiming at producing generalized conclusions. The most critical aspects of PPB-based production and valorisation processes are addressed, including: (i) the identification of the main challenges and potentials of different growth strategies, (ii) a critical analysis of the production of value-added compounds, (iii) a comparison of the different value-added products, (iv) insights into the general challenges and opportunities and (v) recommendations for future research and development towards practical implementation. To date, most of the work has not been executed under real-life conditions, relevant for full-scale application. With the savings in wastewater discharge due to removal of organics, nitrogen and phosphorus as an important economic driver, priorities must go to using PPB-enriched cultures and real waste matrices. The costs associated with artificial illumination, followed by centrifugal harvesting/dewatering and drying, are estimated to be 1.9, 0.3–2.2 and 0.1–0.3 $·kgdry biomass−1. At present, these costs are likely to exceed revenues. Future research efforts must be carried out outdoors, using sunlight as energy source. The growth of bulk biomass on relatively clean wastewater streams (e.g. from food processing) and its utilization as a protein-rich feed (e.g. to replace fishmeal, 1.5–2.0 $·kg−1) appears as a promising valorisation route.

The carrying capacity of the planet is being exceeded, and there is an urgent need to bring forward revolutionary approaches, particularly in terms of energy supply, carbon emissions and nitrogen inputs into the biosphere. Hydrogen gas, generated by means of renewable energy through water electrolysis, can be a platform molecule to drive the future bioeconomy and electrification in the 21st century. The potential to use hydrogen gas in microbial metabolic processes is highly versatile, and this opens a broad range of opportunities for novel biotechnological developments and applications. A first approach concerns the central role of hydrogen gas in the production of bio-based building block chemicals using the methane route, thus, bypassing the inherent low economic value of methane towards higher-value products. Second, hydrogen gas can serve as a key carbon-neutral source to produce third-generation proteins, i.e. microbial protein for food applications, whilst simultaneously enabling carbon capture and nutrient recovery, directly at their point of emission. Combining both approaches to deal with the intermittent nature of renewable energy sources maximises the ability for efficient use of renewable resources.

The transition to sustainable agriculture and horticulture is a societal challenge of global importance. Fertilization with a minimum impact on the environment can facilitate this. Organic fertilizers can play an important role, given their typical release pattern and production through resource recovery. Microbial fertilizers (MFs) constitute an emerging class of organic fertilizers and consist of dried microbial biomass, for instance produced on effluents from the food and beverage industry. In this study, three groups of organisms were tested as MFs: a high‐rate consortium aerobic bacteria (CAB), the microalga Arthrospira platensis (‘Spirulina’) and a purple non‐sulfur bacterium (PNSB) Rhodobacter sp. During storage as dry products, the MFs showed light hygroscopic activity, but the mineral and organic fractions remained stable over a storage period of 91 days. For biological tests, a reference organic fertilizer (ROF) was used as positive control, and a commercial organic growing medium (GM) as substrate. The mineralization patterns without and with plants were similar for all MFs and ROF, with more than 70% of the organic nitrogen mineralized in 77 days. In a first fertilization trial with parsley, all MFs showed equal performance compared to ROF, and the plant fresh weight was even higher with CAB fertilization. CAB was subsequently used in a follow‐up trial with petunia and resulted in elevated plant height, comparable chlorophyll content and a higher amount of flowers compared to ROF. Finally, a cost estimation for packed GM with supplemented fertilizer indicated that CAB and a blend of CAB/PNSB (85%/15%) were most cost competitive, with an increase of 6% and 7% in cost compared to ROF. In conclusion, as bio‐based fertilizers, MFs have the potential to contribute to sustainable plant nutrition, performing as good as a commercially available organic fertilizer, and to a circular economy.

Wastewater treatment plants (WWTPs) have been identified as confirmed but until today underestimated sources of Legionella, playing an important role in local and community cases and outbreaks of Legionnaires’ disease. In general, aerobic biological systems provide an optimum environment for the growth of Legionella due to high organic nitrogen and oxygen concentrations, ideal temperatures and the presence of protozoa. However, few studies have investigated the occurrence of Legionella in WWTPs, and many questions in regards to the interacting factors that promote the proliferation and persistence of Legionella in these treatment systems are still unanswered. This critical review summarizes the current knowledge about Legionella in municipal and industrial WWTPs, the conditions that might support their growth, as well as control strategies that have been applied. Furthermore, an overview of current quantification methods, guidelines and health risks associated with Legionella in reclaimed wastewater is also discussed in depth. A better understanding of the conditions promoting the occurrence of Legionella in WWTPs will contribute to the development of improved wastewater treatment technologies and/or innovative mitigation approaches to minimize future Legionella outbreaks.

To adhere to the Paris Agreement of 2015, we need to store several Gigatonnes (Gt) of carbon annually. In the last years, a variety of technologies for carbon capture and storage (CCS) and carbon capture and usage (CCU) have been demonstrated. While conventional CCS and CCU are techno-economically feasible, their climate change mitigation potentials are limited, due to limited amount of CO2 that can be captured. Hence, there is an urgent need to explore other CCS and CCU routes. Here we discuss an interesting alternative route for capture of carbon dioxide from industrial point sources, using CO2-binding, so-called autotrophic aerobic bacteria to produce microbial biomass as a C-storage product. The produced microbial biomass is often referred to as microbial protein (MP) because it has a crude protein content of ~70–75%. Depending on the industrial production process and final quality of the produced MP, it can be used for human consumption as meat replacement, protein supplement in animal diets, or slow-release organic fertilizer thus providing both organic nitrogen and carbon to agricultural soils. Here, we discuss the potentials and limitations of this so far unexplored CCU approach. A preliminary assessment of the economic feasibility of the different routes for CO2 carbon avoidance, capture and utilization indicates that the value chain to food is becoming attractive and that the other end-points warrant close monitoring over the coming years.

One of the main challenges for the 21st century is to balance the increasing demand for high-quality proteins while mitigating environmental impacts. In particular, cropland-based production of protein-rich animal feed for livestock rearing results in large-scale agricultural land-expansion, nitrogen pollution, and greenhouse gas emissions. Here we propose and analyze the long-term potential of alternative animal feed supply routes based on industrial production of microbial proteins (MP). Our analysis reveals that by 2050, MP can replace, depending on socio-economic development and MP production pathways, between 10-19% of conventional crop-based animal feed protein demand. As a result, global cropland area, global nitrogen losses from croplands and agricultural greenhouse gas emissions can be decreased by 6% (0-13%), 8% (-3-8%), and 7% (-6-9%), respectively. Interestingly, the technology to industrially produce MP at competitive costs is directly accessible for implementation and has the potential to cause a major structural change in the agro-food system.

The Urgent Need to Re-engineer Nitrogen-Efficient Food Production for the Planet

Chapter in: Hülsmann, S., Ardakanian, R. (eds) Managing Water, Soil and Waste Resources to Achieve Sustainable Development Goals. Springer, Cham.

One of the major “sustainability challenges” is to manage the unprecedented demands on agriculture and natural resources to match the increasing human population and consumption of nutritious protein and calories, while dramatically decreasing the environmental footprint in order to maintain the resilience of our planet. Global nitrogen pollution is of particular concern and is already beyond the Earth system’s safe operating space. To meet the world’s future food security, food production needs to be doubled by 2050 and as such will result in further increasing human pressure on the global nitrogen cycle. We argue that there is an urgent need for re-engineering of the anthropogenic nitrogen cycle in order to find a long-term sustainable solution. Firstly, the massive production of plant protein to be upgraded to animal protein has a far too heavy water and land-use footprint to be sustainable. It seriously threatens our freshwater resources by inducing harmful algal blooms through inefficient nutrient use. Secondly, it leads to large scale deforestation in biodiversity hotspots such as the Amazon and Sub-Saharan Africa. Third, the current production chain of plant and animal protein depends strongly on the implementation not only of fertilisers but also of pesticides, pharmaceuticals (e.g., antibiotics), and disinfectants, which indirectly are documented to create phenomena such as multiple antibiotic-resistant bacteria and lower immunological defence and the presence and accumulation of antibiotic-resistant bacteria in agricultural soils. We argue that the line of direct protein production as animal feed or even for human consumption by using microorganisms is a welcome opportunity to alleviate the very significant burden that the contemporary food production systems have on our planet.

The 'microbiome' has become a buzzword. Multiple new technologies allow to gather information about microbial communities as they evolve under stable and variable environmental conditions. The challenge of the next decade will be to develop strategies to compose and manage microbiomes. Here, key aspects are considered that will be of crucial importance for future microbial technological developments. First, the need to deal not only with genotypes but also particularly with phenotypes is addressed. Microbial technologies are often highly dependent on specific core organisms to obtain the desired process outcome. Hence, it is essential to combine omics data with phenotypic information to invoke and control specific phenotypes in the microbiome. Second, the development and application of synthetic microbiomes is evaluated. The central importance of the core species is a no-brainer, but the implementation of proper satellite species is an important route to explore. Overall, for the next decade, microbiome research should no longer almost exclusively focus on its capacity to degrade and dissipate but rather on its remarkable capability to capture disordered components and upgrade them into high-value microbial products. These products can become valuable commodities in the cyclic economy, as reflected in the case of 'reversed sanitation', which is introduced here.

The microbiome as engineering tool: Manufacturing and trading between microorganisms

New Biotechnology, Volume 39, Part B, 25 October 2017, Pages 206-214

The integration of microbial technologies within the framework of the water-energy nexus has been taking place for over a century, but these mixed microbial communities are considered hard to deal with ‘black boxes’. Process steering is mainly based on avoiding process failure by monitoring conventional parameters, e.g., pH and temperature, which often leads to operation far below the intrinsic potential. Mixed microbial communities do not reflect a randomised individual mix, but an interacting microbiological entity. Advance monitoring to obtain effective engineering of the microbiome is achievable, and even crucial to obtain the desired performance and products. This can be achieved via a top-down or bottom-up approach. The top-down strategy is reflected in the microbial resource management concept that considers the microbial community as a well-structured network. This network can be monitored by means of molecular techniques that will allow the development of accurate and quick decision tools. In contrast, the bottom-up approach makes use of synthetic cultures that can be composed starting from defined axenic cultures, based on the requirements of the process under consideration. The success of both approaches depends on real-time monitoring and control. Of particular importance is the necessity to identify and characterise the key players in the process. These key players not only relate with the establishment of functional conversions, but also with the interaction between partner bacteria. This emphasises the importance of molecular (screening) techniques to obtain structural and functional insights, minimise energy input, and maximise product output by means of integrated microbiome processes.

Spent sulphite liquor (SSL) was used as carbon source for the production of succinic acid using immobilized cultures of Actinobacillus succinogenes and Basfia succiniciproducens on two different supports, delignified cellulosic material (DCM) and alginate beads. Fed-batch immobilized cultures with A. succinogenes in alginates resulted in higher sugar to succinic acid conversion yield (0.81 g/g) than the respective yield achieved (0.65 g/g) when DCM immobilized cultures were used. The final succinic acid concentration and yield achieved in fed-batch with immobilized cultures of B. succiniciproducens in alginates (45 g/L and 0.66 g/g) were higher than A. succinogenes immobilized cultures (35.4 g/L and 0.61 g/g) using nano-filtrated SSL as fermentation medium. Immobilized cultures of B. succiniciproducens in alginate beads were reused in four sequential fed-batch fermentations of nano-filtrated SSL leading to the production of 64.7 g of succinic acid with a yield range of 0.42–0.67 g/g and productivity range of 0.29–0.65 g/L/h. The immobilized cultures improved the efficiency of succinic acid production as compared to free cell cultures.

Microbes and the Next Nitrogen Revolution

Environ. Sci. Technol. 2017, 51, 13, 7297-7303

The Haber Bosch process is among the greatest inventions of the 20th century. It provided agriculture with reactive nitrogen and ultimately mankind with nourishment for a population of 7 billion people. However, the present agricultural practice of growing crops for animal production and human food constitutes a major threat to the sustainability of the planet in terms of reactive nitrogen pollution. In view of the shortage of directly feasible and cost-effective measures to avoid these planetary nitrogen burdens and the necessity to remediate this problem, we foresee the absolute need for and expect a revolution in the use of microbes as a source of protein. Bypassing land-based agriculture through direct use of Haber Bosch produced nitrogen for reactor-based production of microbial protein can be an inspiring concept for the production of high quality animal feed and even straightforward supply of proteinaceous products for human food, without significant nitrogen losses to the environment and without the need for genetic engineering to safeguard feed and food supply for the generations to come.

Domestic used water treatment systems are currently predominantly based on conventional resource inefficient treatment processes. While resource recovery is gaining momentum it lacks high value end-products which can be efficiently marketed. Microbial protein production offers a valid and promising alternative by upgrading low value recovered resources into high quality feed and also food. In the present study, we evaluated the potential of hydrogen-oxidizing bacteria to upgrade ammonium and carbon dioxide under autotrophic growth conditions. The enrichment of a generic microbial community and the implementation of different culture conditions (sequenced batch resp. continuous reactor) revealed surprising features. At low selection pressure (i.e. under sequenced batch culture at high solid retention time), a very diverse microbiome with an important presence of predatory Bdellovibrio spp. was observed. The microbial culture which evolved under high rate selection pressure (i.e. dilution rate D = 0.1 h−1) under continuous reactor conditions was dominated by Sulfuricurvum spp. and a highly stable and efficient process in terms of N and C uptake, biomass yield and volumetric productivity was attained. Under continuous culture conditions the maximum yield obtained was 0.29 g cell dry weight per gram chemical oxygen demand equivalent of hydrogen, whereas the maximum volumetric loading rate peaked 0.41 g cell dry weight per litre per hour at a protein content of 71%. Finally, the microbial protein produced was of high nutritive quality in terms of essential amino acids content and can be a suitable substitute for conventional feed sources such as fishmeal or soybean meal.

Ultrafiltration and nanofiltration of spent sulphite liquor (SSL) has been employed to evaluate the simultaneous production of lignosulphonates and bio-based succinic acid using the bacterial strains Actinobacillus succinogenes and Basfia succiniciproducens. Ultrafiltration with membranes of 10, 5 and 3kDa molecular weight cut-off results in significant losses of lignosulphonates (26-50%) in the permeate stream, while nanofiltration using membrane with 500Da molecular weight cut-off results in high retention yields of lignosulphonates (95.6%) in the retentate stream. Fed-batch bioreactor cultures using permeates from ultrafiltrated SSL resulted in similar succinic acid concentration (27.5g/L) and productivity (0.4g/L/h) by both strains. When permeates from nanofiltrated SSL were used, the strain B. succiniciproducens showed the highest succinic acid concentration (33.8g/L), yield (0.58g per g of consumed sugars) and productivity (0.48g/L/h). The nanofiltration of 1t of thick spent sulphite liquor could lead to the production of 306.3kg of lignosulphonates and 52.7kg of succinic acid, whereas the ultrafiltration of 1t of thick spent sulphite liquor using a 3kDa membrane could result in the production of 237kg of lignosulphonates and 71.8kg of succinic acid when B. succiniproducens is used in both cases.

Microbial biotechnology has a long history of producing feeds and foods. The key feature of today's market economy is that protein production by conventional agriculture based food supply chains is becoming a major issue in terms of global environmental pollution such as diffuse nutrient and greenhouse gas emissions, land use and water footprint. Time has come to re-assess the current potentials of producing protein-rich feed or food additives in the form of algae, yeasts, fungi and plain bacterial cellular biomass, producible with a lower environmental footprint compared with other plant or animal-based alternatives. A major driver is the need to no longer disintegrate but rather upgrade a variety of low-value organic and inorganic side streams in our current non-cyclic economy. In this context, microbial bioconversions of such valuable matters to nutritive microbial cells and cell components are a powerful asset. The worldwide market of animal protein is of the order of several hundred million tons per year, that of plant protein several billion tons of protein per year; hence, the expansion of the production of microbial protein does not pose disruptive challenges towards the process of the latter. Besides protein as nutritive compounds, also other cellular components such as lipids (single cell oil), polyhydroxybuthyrate, exopolymeric saccharides, carotenoids, ectorines, (pro)vitamins and essential amino acids can be of value for the growing domain of novel nutrition. In order for microbial protein as feed or food to become a major and sustainable alternative, addressing the challenges of creating awareness and achieving public and broader regulatory acceptance are real and need to be addressed with care and expedience.

Spent sulphite liquor (SSL) has been used for the production of lignosulphonates (LS), antioxidants and bio-based succinic acid. Solvent extraction of SSL with isopropanol led to the separation of approximately 80% of the total LS content, whereas the fermentations carried out using the pretreated SSL with isopropanol led to the production of around 19 g/L of succinic acid by both Actinobacillus succinogenes and Basfia succiniciproducens. Fractionation of SSL via nanofiltration to separate the LS and solvent extraction using ethyl acetate to separate the phenolic compounds produced a detoxified sugar-rich stream that led to the production of 39 g/L of succinic acid by B. succiniciproducens. This fractionation scheme resulted also in the production of 32.4 g LS and 1.15 g phenolic-rich extract per 100 g of SSL. Both pretreatment schemes separated significant quantities of metals and heavy metals. This novel biorefinery concept could be integrated in acidic sulphite pulping mills.

Microbial healing of concrete cracks is a relatively slow process, and meanwhile the steel rebar is exposed to corrosive substances. Nitrate reducing bacteria can inhibit corrosion and provide crack healing, by simultaneously producing NO2− and inducing CaCO3 precipitation. In this study, the functionality of one non-axenic and two axenic NO3− reducing cultures for the development of corrosion resistant self-healing concrete was investigated. Both axenic cultures survived in mortar when incorporated in protective carriers and became active 3 days after the pH dropped below 10. The non-axenic culture named “activated compact denitrifying core” (ACDC) revealed comparable resuscitation performance without any additional protection. Moreover, ACDC induced passivation of the steel in corrosive electrolyte solution (0.05 M NaCl) by producing 57 mM NO2− in 1 week. The axenic cultures produced NO2− up to 26.8 mM, and passivation breakdown and pitting corrosion were observed. Overall, ACDC appears suitable for corrosion resistant microbial self-healing concrete.

Production of non-axenic ureolytic spores for self-healing concrete applications

Construction and Building Materials, Volume 93, 15 September 2015, Pages 1034-1041

The bio-based self-healing concrete market demands an inexpensive bio-agent. The use of axenic ureolytic spore cultures has been demonstrated to be efficient but too expensive, due to an operational expense (OPEX) cost of about 500 €/kg of bio-agent. A new selection process to obtain a powderous material containing an efficient ureolytic microbial community (Cyclic EnRiched Ureolytic Powder or CERUP) has been developed. Ureolytic activity, calcium carbonate precipitation capability and the effects in concrete were evaluated at production scales of 5 L and 50 L. The non-axenic culture obtained following this new selective process, at both 5 L and 50 L scales, proved to be as good as the benchmark Bacillus sphaericus both in urea hydrolysis (20 g urea/L in 24 h) and calcium carbonate precipitation (0.3 g CaCO3/g VS.h). Plain incorporation of CERUP in concrete was found to be efficient at levels of 0.5% and 1% of the cement weight. Furthermore, a brief economical evaluation was performed to verify the economic feasibility of this product. Only OPEX costs were considered since capital expense (CAPEX) costs are directly related to the dimensions of scale and thus not possible to estimate at this stage of the research. The OPEX cost per unit of CERUP is about 40 times lower than the OPEX cost of a B. sphaericus axenic culture. However, even with such decrease in cost, the production of bacterial spores to incorporate in concrete is too expensive.

The increase in the world population, vulnerability of conventional crop production to climate change, and population shifts to megacities justify a re-examination of current methods of converting reactive nitrogen to dinitrogen gas in sewage and waste treatment plants. Indeed, by up-grading treatment plants to factories in which the incoming materials are first deconstructed to units such as ammonia, carbon dioxide and clean minerals, one can implement a highly intensive and efficient microbial resynthesis process in which the used nitrogen is harvested as microbial protein (at efficiencies close to 100%). This can be used for animal feed and food purposes. The technology for recovery of reactive nitrogen as microbial protein is available but a change of mindset needs to be achieved to make such recovery acceptable.

152 page minimum spine. Editor: Matt Hotze, JEM: Esther
RTP:  Diane Murphy

Screening of bacteria and concrete compatible protection materials

Construction and Building Materials, Volume 88, 30 July 2015, Pages 196-203

Bacteria that can induce calcium carbonate precipitation have been studied for self-healing concrete applications. Due to the harsh environment of concrete, i.e. very high pH, small pore size and dry conditions, protection methods/materials have been used to preserve the bacterial agents. A wide screening of commercially available materials is thus required to evaluate them as alternatives. This study describes the influence of six commercially available possible protection approaches (diatomaceous earth, metakaolin, expanded clay, granular activated carbon, zeolite and air entrainment) on mortar setting and compressive strength when combined with either Bacillus sphaericus spores or Diaphorobacter nitroreducens and their respective nutrients. The influence of two novel, self-protected, bacterial agents was also investigated within the same scope. The most severe effect on setting time was observed as an undesirable delay of 340 min in all samples containing nutrients for ureolytic bacteria. Samples containing B. sphaericus spores showed the most significant decreases in compressive strength up to 68%. Yet, the addition of either D. nitroreducens or its respective nutrients did not cause major impact on both the setting times and the compressive strengths of the mortar specimens. The latter thus appears to be a suitable bacterial agent for further research on self-healing concrete. Likewise, the use of the novel self-protected bacterial agents did not affect the setting and the compressive strength of mortar. These results pave the way to replace protection materials with self-protection techniques. The latter should be further investigated for development of microbial self-healing concrete.

Resources in used water are at present mainly destroyed rather than reused. Recovered nutrients can serve as raw material for the sustainable production of high value bio-products. The concept of using hydrogen and oxygen, produced by green or off-peak energy by electrolysis, as well as the unique capability of autotrophic hydrogen oxidizing bacteria to upgrade nitrogen and minerals into valuable microbial biomass, is proposed. Both axenic and mixed microbial cultures can thus be of value to implement re-synthesis of recovered nutrients in biomolecules. This process can become a major line in the sustainable “water factory” of the future.

There is a growing demand for silver-based biocides, including both ionic silver forms and metallic nanosilver. The use of metallic nanosilver, typically chemically produced, faces challenges including particle agglomeration, high costs, and upscaling difficulties . Additionally, there exists a need for the development of a more eco-friendly production of nanosilver. In this study, Gram-positive and Gram-negative bacteria were utilized in the non-enzymatic production of silver nanoparticles via the interaction of silver ions and organic compounds present on the bacterial cell. Only lactic acid bacteria, Lactobacillus spp., Pediococcus pentosaceusEnterococcus faecium, and Lactococcus garvieae, were able to reduce silver. The nanoparticles of the five best producing Lactobacillus spp. were examined more into detail with transmission electron microscopy. Particle localization inside the cell, the mean particle size, and size distribution were species dependent, with Lactobacillus fermentum having the smallest mean particle size of 11.2 nm, the most narrow size distribution, and most nanoparticles associated with the outside of the cells. Furthermore, influence of pH on the reduction process was investigated. With increasing pH, silver recovery increased as well as the reduction rate as indicated by UV–VIS analyses. This study demonstrated that Lactobacillus spp. can be used for a rapid and efficient production of silver nanoparticles.

The effect of three different types of glycerol on the performance of up-flow anaerobic sludge blanket (UASB) reactors treating potato processing wastewater was investigated. High COD removal efficiencies were obtained in both control and supplemented UASB reactors (around 85%). By adding 2 ml glycerol product per liter of raw wastewater, the biogas production could be increased by 0.74 l biogas ml−1 glycerol product, which leads to energy values in the range of 810–1270 kWhelectric per m3 product. Moreover, a better in-reactor biomass yield was observed for the supplemented UASB reactor (0.012 g VSS g−1 CODremoved) compared to the UASB control (0.002 g VSS g−1 CODremoved), which suggests a positive effect of glycerol on the sludge blanket growth.

A new approach for the removal of the pesticide lindane (γ-hexachlorocyclohexane or γ-HCH) makes use of catalytic reduction of HCH to benzene over a metal catalyst, namely Pd(0). Since specific surface area plays an important role in reactivity of catalysts, this study investigated the use of bioPd(0), i.e. nano-scale Pd(0) particles precipitated on the biomass of Shewanella oneidensis, for the removal of lindane. It was demonstrated that bioPd(0) has catalytic activity towards dechlorination of γ-HCH, with the addition of formate as electron donor, and that dechlorination with bioPd(0) was more efficient than with commercial powdered Pd(0). The biodegradable compound benzene was formed as reaction product and other HCH isomers could also be dechlorinated. Subsequently bioPd(0) was implemented in a membrane reactor technology for the treatment of γ-HCH polluted water. In a fed-batch process configuration with formate as electron donor, a removal percentage of 98% of γ-HCH saturated water (10 mg l−1) was achieved within 24 h. The measured chloride mass balance approached the theoretical value. The results of this work showed that a complete, efficient and fast removal of lindane was achieved by biocatalysis with bioPd(0).

The transport and activity of Desulfitobacterium dichloroeliminans strain DCA1 in 1,2-dichloroethane (1,2-DCA)-contaminated groundwater have been evaluated through an in situ bioaugmentation test at an industrial site (Belgium). The migration of strain DCA1 was monitored from an injection well toward a monitoring well, and the effect of the imposed groundwater flow on its distribution was assessed by means of transport model MOCDENS3D. The results of the real-time PCR (16S rRNA gene) quantification downstream from the injection point were used to evaluate the bacterial distribution pattern simulated by MOCDENS3D. In the injection well, the 1,2-DCA concentration in the groundwater decreased from 939.8 to 0.9 μM in a 35 day time interval and in the presence of a sodium lactate solution. Moreover, analyses from the monitoring well showed that the cells were still active after transport through the aquifer, although biodegradation occurred to a lesser extent. This study showed that strain DCA1 can be successfully applied for the removal of 1,2-DCA under field conditions and that its limited retardation offers perspectives for large-scale cleanup processes of industrial sites.

Quantifying microorganisms responsible for bioremediation can provide insight in their behavior and can help to obtain a better understanding of the physicochemical parameters monitored during bioremediation. A real time PCR (RTm PCR) assay based on the detection with SYBR Green I was optimized in order to quantify the 1,2-dichloroethane dehalorespiring Desulfitobacterium dichloroeliminans strain DCA1. A primer pair targeting unique regions of the 16 S rRNA gene was designed and tested in silico for its specificity. Selectivity was furthermore evaluated and a Limit of Quantification of 1.5·104 cells/μL DNA extract was obtained for spiked groundwater. Real time measurements of groundwater samples retrieved from a bioaugmented monitoring well and which had an average concentration lying in the range of the Limit of Quantification were evaluated positively with regards to reproducibility. Validation of the RTm PCR assay on groundwater samples originating from different sites confirmed the specificity of the designed primer pair. This RTm PCR assay can be used to survey the abundance and kinetics of strain DCA1 in in situ bioaugmentation field studies.

The total ammonium nitrogen (TAN) concentration is often a key limiting water quality parameter in intensive aquaculture systems. Removing ammonia (NH3) through biological activity is thus an important objective in aquaria and aquaculture system designs. In this study, the performance characteristics of a suspension of nitrifying cells (named ammonia binding inoculum liquid, ABIL) have been explored. This aqueous suspension contains a highly active, nitrifying microbial consortium that can be used to shorten the start-up period of a biofilter.

Tests were performed in freshwater at lab scale (70 l, 20–24 °C). Results showed that the application of the consortium at a dose of 5 mg volatile suspended solids (VSS) l−1 assures a total removal of ammonium (NH4+) and nitrite species from 10 mg N l−1 to below the detection limit within a period of 4 days. Experimentally, at a substrate level of 10 mg TAN l−1, a rate of biological ammonium and nitrite conversion of the order 0.3–0.5 g TAN g−1 VSS−1 day−1 could be achieved by the consortium in the freshwater aquaria systems tested. Provided adequate aeration and dissolved oxygen (DO) levels of 6 mg l−1 or more, no important intermediary nitrite concentrations were found. Only a small amount of TAN was not recovered as nitrate and might have been lost through ammonia stripping. Pre-inoculating the nitrifiers in polyurethane (PU) sponges and installation of such sponges in the freshwater aquaria did not improve the effect compared to adding the consortium directly to the water.

After 12 months preservation of the inoculum at 4 °C, no important decrease in ammonium removal activity and only a minor decrease in the nitrite removal rate of the consortium were noticed.

Improvement of activated sludge performance by the addition of Nutriflok 50 S

Water Research, Volume 31, Issue 2, February 1997, Pages 366-371

By means of rigorously controlled lab-scale activated sludge systems operated in parallel, the effect of the addition of a nutritive supplement on the overall performance of these systems was verified. Nutriflok 50 S is a commercially available product, containing nutrients and flocculating agents. In a first series of tests by means of SCAS (semi-continuous activated sludge) reactors, fed with synthetic wastewater, the positive effect of the supplement (dosed at 5% of the chemical oxygen demand (COD) load) on the sludge volume index (SVI) (39% improvement) and the residual COD (40% improvement) was demonstrated. In a second series, an industrial wastewater was used in a benchscale set-up according to the OECD procedure. The overall efficiency and sludge sedimentation characteristics were compared for two parallel activated sludge systems continuously fed with pharmaceutical wastewater. In the control no additive was given; the other reactor received on a daily basis a dose of Nutriflok 50 S corresponding to 10% of the COD load. The performance of the Nutriflok 50 S supplemented reactor was found to be statistically better in terms of sedimentation characteristics, nitrification and COD removal than the control. Especially when operated under winter conditions (10°C) and at higher sludge loading rates (Bx = 0.26 kg COD/kg VSS.d) the COD removal efficiency was about 35% higher in the treated reactor. Moreover, nitrification could be maintained in the supplemented reactor at low temperatures, whilst it became minimal under those conditions in the control reactor. Also in the second series of tests, sludge sedimentation characteristics improved with Nutriflok 50 S. The SVI was 40% lower for the Nutriflok 50 S amended sludge and the recycled sludge was 27% more concentrated. When operated at higher temperatures (20–26°C) the amended sludge was less sensitive to denitrification linked flotation in the settler. Microscopic examination of the sludge revealed a more diverse microbial community and the presence of higher trophic organisms in the supplemented reactor. These results indicate a potential for considerable improvement, not only of the overall conversion efficiency, but also of the sludge management characteristics when Nutriflok 50 S is dosed to the activated sludge.

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