Merah Mas Blog

A PhD report that could just be my proudest achievement to date has just been submitted. This is the closing remarks section. The Executive Summary was posted in a previous post. If you want the full report, please let me know.

8. CLOSING REMARKS

The concept of industrial metabolism requires the maximisation of resource productivity by ensuring that all resources exploited provide the maximum possible products and services. This approach is a key premise to achieving sustainable systems for our society and minimising the environmental burden associated with anthropogenic acitivity. In addressing this, it is valuable to view wastes, including wastewater, as a potential raw material resource. This approach can be used through the wastewater biorefinery concept which is centred on two major outcomes: the treatment of wastewater to the desired water quality and the simultaneous production of products of value, as either commodity or energy products. Unit operations in such a wastewater biorefinery include reactors that can reduce nutrient loads in wastewater in a variety of environments, while producing a range of valuable bioproducts that can potentially sustain jobs, meet commodity needs and fund capacity building through their revenue. This review addresses the requirements of such reactor systems in which microbial bioprocesses are used for the production of commodity polymers while reducing nutrient loads. Because of the non-sterile nature of the wastewater environment, it is best to select micro-organisms for product formation that also fulfill a role in the microbial ecology and culture conditions and product which contribute a selective advantage to the microbial population. Hence, wastewater biorefineries are not suitable for all bioproducts. The overall project considers this application in terms of the model system in which microbial communities enriched for Bacillus species are used for the production of poly-glutamic acid.

Poly-glutamic acid (PGA) is hypothesized to fulfil an ecological role to give Bacillus (or other species producing PGA) a competitive advantage that can be exploited for bioproduction through microbial community engineering in wastewater biorefineries. The reactor design is crucial to achieve this microbial community engineering. In order to achieve the production of relatively pure, N-rich PGA from wastewater, the following need to be addressed:

• Establishment of Bacillus as the dominant organism in the mixed microbial community used for PGA production;
• Determining a reactor configuration suited to retention of biomass while processing large volumes of dilute wastewater;
• Selection of a downstream process train that takes cognisance of the dilute product environment, and intercalates with the reactor system in the optimal manner;
• Selection of a reactor configuration and associated downstream processing unit operations that are appropriate for the production of a low value product in dilute solution.

Two reactor designs have been highlighted that are proposed to fulfill this function as well as contribute to effective product recovery: the Aerobic Granular Sludge (AGS) process, and the HYbrid Bacillus ACtivated Sludge (HYBACS) process.

The AGS process is defined by the characteristics of the sludge particles. The granules are defined as “aggregates of microbial origin, which do not coagulate under reduced hydrodynamic shear (‘sludge bulking’) and which settle significantly faster than activated sludge flocs” (15 sec vs 20 min) (de Kreuk et al 2007a).

The HYbrid Bacillus ACtivated Sludge (HYBACS) process is a modified Rotating Biological Contactor (RBC) system. RBC’s are non-submerged, attached growth bioreactors, similar to trickling filters, with circular media mounted (approx 3.6m in diameter for standard units) on a horizontal shaft, partially submerged (typically 40%) in the wastewater, and rotated at a speed of one to six revolutions per minute (Grady et al 2011). The circular support media is modified to form a high surface area mesh disc to support an active biofilm.

These reactors were selected for detailed discussion because the management of biofilm detachment – and hence product recovery – are controllable. The support material of the HYBACS is relatively planar, while the granular system does not have a carrier, with settling properties that make product recovery possible. In addition, the biofilm zonation in both reactor types (allowing for heterotrophy, nitrification, phosphate removal and denitrification zones intrinsic in the biofilm) is advantageous.

The AGS process is operated as a Sequencing Batch Reactor (SBR). It is a time-oriented process that can be designed and operated to simulate virtually all conventional continuous-flow activated sludge systems, from contact stabilization to extended aeration, making it a useful approach in a laboratory environment. This aspect, combined with the rapid sedimentation velocity possible with aerobic granules allows product formation and downstream processing to form part of the reactor design at discrete steps (Johnson, 2010).

The HYBACS process can accommodate a wider range of substrate loads and is a simpler process to operate than the AGS process. The combination of the meshed internal structure and planar disk macro structure gives product recovery options that compliment the AGS process, and the overal operation of the HYBACS promotes Bacillus dominance.

PGA is generally accepted to be a product excreted into the bulk solution and not even weakly associated with the biomass. Several biofilm-based approaches allow cultivation at relatively low flow rates and may be used to minimise the separation of the weakly associated polymer from the biomass. In terms of considering PGA recovery, three potential scenarios have to be considered:

1. PGA is excreted into the bulk solution and is not associated with the biomass at all
2. PGA is excreted, but weakly associated with the biomass. This association is easily disrupted
3. PGA is excreted, but strongly associated with the biomass. Significant physical or chemical means need to be employed to disrupt this association.

The AGS and HYBACS processes will have different utility towards PGA product recovery depending on which of these scenarios come into play. Scenario 1 and 2 are more suited to the HYBACS, while scenario 3 favours the AGS process.

This work carries significance in arguing for a different approach to waste, and serves to introduce the discussion on how to use significant infrastructural limitations to the advantage of wastewater biorefineries. Challenges in optimising the system rather than individual unit processes are highlighted, which include the different hegemonies present in the interface between environmental and bioprocess engineering disciplines. This review particularly considers the aspects to consider when reactors are designed as unit processes in a wastewater biorefinery, and contributes to a better understanding of reactor selection in areas where systems are required to meet the needs of biomass retention, processing of large volumes and integrated product recovery.

This work has broader applicability by complementing current research to understand and better control erosion and abrasion while minimising sloughing (considered in this project as product recovery). The remaining challenge in the engineering of these wastewater biorefinery systems, as with the wider body of work on biofilm processes is directed towards the control of biofilm structure and morphology, which depends on, and at the same time affects, the reactor hydrodynamics and mass transfer characteristics.

1. EXECUTIVE SUMMARY

This review discusses bioreactor considerations to leverage the value to be obtained from waste. Section 2 gives a general introduction to the wider project, which takes lessons from both environmental and bioprocess engineering. This project is concerned with proving the technology to produce valuable products from dilute waste streams, and less concerned about the performance and application of the produced material. Reactor design is the main area that can be optimized for bioproduction, to place greater emphasis on the optimal functioning of the system as a whole. The energy utilisation of reactors needs to be improved, and the reactor needs to be designed with downstream processing in mind.

Section 3 introduces the reader to the concept of wastewater biorefineries. The wastewater biorefinery is established to maximize biomass productivity by ensuring that, not only is the wastewater treated to the necessary standard (yielding the outgoing water product), but that components removed from this wastewater are converted to products of value (economically or socially or both). The concept of a wastewater biorefinery is built around the overarching ability to function in the face of significant infrastructural limitations and, through its operation, improve these limitations.

Wastewater biorefineries move away from the traditional wastewater treatment objectives. Section 4 discusses how the system objectives change, and are affected by the differences in approach between the different stakeholders involved in their design and operation. From a process point of view multiple aspects need to be considered, including

• Efficient product recovery of both the bioproduct and water product;

• Energy efficiency through utilising both biomass retention and product retention;

• The ability to be retrofitted into existing plants;

• The implications of maintaining product productivity in non-sterile, mixed, cultures.

The social implications that arise when a new discipline emerges needs to be considered. These develop from disciplines that differ in their approach to reactor design, the objectives that the reactor is designed for and the prevalent culture and values. Emerging disciplines often co-incide with changing generations, which influence the preferred technologies used and ease of adapting to more advanced forms of analysis. The different backgrounds in especially the younger generation often leads to tensions, where bioprocess engineers often have a stronger scientific background, which leads to a different professional discourse. As the field of wastewater biorefineries develops its own language and discourse, supportive technologies mature and the boundaries between the parent biotechnologies are reduced, development of new design parameters and the development of new design approaches will occur, leading to a new generation of professionals. Section 4.3 highlights some of these tensions in the research environment, where data obtained in the laboratory for bioprocessing purposes often cannot be applied directly to wastewater bioprocesses. Section 4 also highlights emergent phenomena that arises when existing systems are adapted for use that they were not originally intended for.

Section 5 illustrates the suitability of Bacillus as a production organism in a mixed culture, specifically in its capacity to produce polyglutamic acid (PGA). Section 5.1 gives a brief introduction to the widespread use of Bacillus in industry. PGA’s role in serving an ecological function is highlighted, which supports its value towards contributing to Bacillus dominance and hence higher potential productivity. The rationale of this project is illustrated by highlighting the uses of PGA in industry, most notably that of bioflocculant and soil conditioner. Section 5.4 argues for the production of PGA in wastewater biorefineries by showcasing previous work that produced PGA from waste resources. The value of using Bacillus in wastewater biorefineries is used as an example to show how working with Nature can give overall benefit to both humans and the environment. In Section 5.5 the positive role that Bacillus plays in wastewater treatment is discussed, alluding to the possibility to improve the natural system while providing opportunity for economic gain. Section 5.6 shows how producing PGA, using Bacillus, from a dilute waste can provide benefits over using conventional bioprocessing.

The first half of this review considers which factors are crucial to develop a bioreactor in a wastewater biorefinery. Section 6 provide the required review of reactor systems available to meet the needs of biomass retention, processing of large volumes and integrated product recovery. While not considered in this project, solid substrate fermentation (SSF) reactors are included for its potential in using biosolids to produce valuable products. Based on the combination of the need for biomass retention and the need for product recovery, the two reactors that were selected for further discussion is the aerobic granular sludge (AGS) process, an example of a particle based biofilm reactor (Section 6.6) and a hybrid rotating biological contactor (RBC) based on the HYbrid Bacillus ACtivated Sludge (HYBACS) process (Section 6.7). Scale up will not be considered in this project, but where possible the relevent calculations and data will be included to develop scale-up effectively and build confidence in reactor types as viable alternatives to the stirred tank reactor (STR). Specifically, laboratory data will be generated with an awareness of that required to inform scale-up.

This review focuses on the reactor configurations to produce PGA-containing product to be used for bioflocculant or soil conditioning applications. Downstream processing (DSP) is often only considered after the reactor is designed and separately to it. This may lead to an unoptimised system. To design the reactor system with DSP in mind to facilitate their combined optimisation, a brief overview and proposed DSP options are given in Section 7.

Closing remarks are given in Section 8, and Section 10 lists the numerous acknowledgements, illustrating that with the more transdisciplinary approach required when optimising the system as a whole, more and more players become important. The greatest value in wastewater biorefineries lies not in the technology or adequate engineering design, but in the connections between people, adequate communication and a willingness to engage.

in the forest

After yet another wobbly and oh-my-gosh-what-am-i-doing-here time, throwing me back into dark days that I really didn’t want to go back to. So my pre-new years resolution is to blog about this once a week. Sortof supervising myself  with weekly progress reports (and with the fickle wish that supervision from the world wide web will perhaps emerge).

I have this mental picture of myself running in circles of varying diameter across varying terrain – sometimes in a forest (low light, clueless, but feeling good about it), sometimes in mud (overwhelmed with knowledge but no clue what to do with it), sometimes in a featureless desert (just… nothing, and not sure if this is good or bad) and often into and out of a huge vat of peanut butter (just overwhelmed and stuck and sticky and out of breath). And always, always, alone. I think the PhD is this thin line radiating out of the centre, which means I stumble across it only once in a blue moon.

One thing I occasionally realise is there are huge paradigms that I explore. These are incorporated into TEDx, ShackLabs, Merah Mas etc, but they don’t really have a place in the PhD. So when I try to force them into the PhD everything derails. On the other hand, they inform the PhD, they are the context, the reason it exists. So when I try to sideline them to focus on the PhD, the whole thing falls down as well. The PhD is this wobbly, oscillating tightrope caught in the storms of our changing, breaking world with these exciting things enticing me from below, asking me to let go, but I walk it, crawl it, and most of the time hold on for dear life – with my teeth. On the one side, if I turn around, is mediocrity. On the other, a changed world of my making. If I let go, it’s the definition of hell (be-careful-what-you-wish-for type of vibe).

I decided a while ago to walk this tightrope by blog, if only to chart the course and in later years laugh at it. So here’s another edition.

Before I launch into the TOC (Table of Contents) bit, points that are important in the context of the project (not thought through yet, but these keep popping up. I don’t expect them to make sense yet):

1. I’m looking from an Industrial Symbiosis point of view, not an Ecology. This project is not an analysis, neither is it systems wide. I focus on the business case of a few identified, clearly delineated industry partners, and try to make my technology suitable to partner with them.
2. Industrial symbioses or even ecosystems can not be planned for. They emerge spontaneously, but can be incentivised by appropriate technologies – reference Pierre Desrochers (e.g. Cities and Industrial Symbiosis: Some Historical Perspectives and Policy Implications Journal of Industrial Ecology, DOI: 10.1162/10881980160084024.
3. Informal South/ ShackLabs approach (unlikely to be implemented in this project, but informs design). e.g. environmentally sustainable infrastructure in the informal context – Informal South (Reference Shannon Royden-Turner)
1. Place the design in the context it will function in
2. Be committed to the community within which it will function
3. Collaborate and co-create research and development proposals
4. Appreciate and integrate local knowledge
5. Design for community ownership and maintenance
6. Shift the power of decision making to the people who will be faced with the consequences
7. Work on a Sense-and-Respond model rather than Predict-and-Control
4. Restorative ecology / Restorative redevelopment – ecosystem sevices linking up with economic services.

“A fusion of ecological restoration and sustainable development, restorative redevelopment represents an emerging paradigm for remediating landscapes. Rather than merely fixing the broken bits and pieces of nature, restorative development advocates the reuse of devastated landscapes to improve the value and livability of a location for humans at the same time as effectively reinstating natural processes and functions.” – Robert France, (Restorative Redevelopment of Devastated Ecocultural Landscapes, 2011).

While I use Biomimicry as a design tool, talk about ecosystem metaphors etc, and aim to restore ecosystems, I acknowledge that going back to what was before the intervention occurred may not be sufficient: we are working towards something new, entire new systems that in themselves are unpredictable. Also, nature is not a benevolent god-like force. It’s only more complex that we can understand. It is neither the ultimate goal nor in need of ‘saving’.

Consider the Leverage points to intervene in a system – reference Donella Meadows (Thinking in Systems, 2008)

Working title of PhD: bioproduct production using dilute wastewater and aerobic mixed microbial cultures

I take Katja Johnson’s thesis (TU Delft) from 2010 as a rough guideline, and acknowledge the inspiration her thesis and her promoters (dr Robbert Kleerebezem & Prof Mark van Loosdrecht) gave me:
Chapter 1: Introduction
Chapter 2: Model based evaluation of data
Chapter 3: Influence of C/N ratio
Chapter 4: Short and Long term Temperature Effects
Chapter 5: Influence of Ammonium
Chapter 6: Best Case Scenario
Chapter 7: General Discussion.

keywords: mixed culture biotechnology, non-sterile waste streams, open reactors, polyglutamic acid (PGA), bioproduct, dilute wastewater.

In my thesis I aim to follow this outline with application to PGA (and proposed modifications further explained below), with addition of two aspects;

• product recovery (product is extracellular), and
• effect of reinoculation, or exploiting sporulation to improve Bacillus / PGA-producer dominance.

I also look at two reactor setups, aerobic granular sludge (AGS – affectionately called Agnes), and rotating biological contactors (RBC – or Roberta – similar to a spitbraai for bugs, without the cooking bit). AGS because it is a new technology with multiple benefits relevant to bioproduction, and RBC because of the established Bacillus presence in RBC’s in general, the suitability of the two reactor types to different substrate loading, but mostly because of the product recovery and organism dominance (aka need two reactor designs to cover these two aspects).

I would also like to build in ‘community ownership and management models’ by approaching the reactor system as a Sense and Respond system rather than a Control and Predict system – something that I believe mixed culture biotechnology and microbial community engineering already does. I want to formalise this approach into the Industrial Symbiosis aspects. (I still call it IndieEco (from Industrial Ecology) – but redefine it as the interaction of the INDIvidual with her ECOsystem). This starts building into the ShackLabs approach.

Katja uses a two step process, 1) a culture enrichment and growth step and ii) a PHA production step.
The selective pressure for culture enrichment is through feast and famine phase – as PHA is a storage polymer, and we are selecting for organisms that have a very fast substrate uptake rate, giving them competitive advantage. We can use this same approach for PGA. The substrate uptake rate is decoupled from growth, and this has been found for PGA as well (reference Morikawa, 2006) – but Bacillus PGA production is growth associated in suspended culture and non-growth associated in a biofilm (aka very dilute substrate concentrations). Exploring this is a novel contribution to knowledge. (Looking at PGA per se in this system is also a novel contribution to knowledge)
This also has implications for the kinetic model developed by Margaritis et al (2004).

Coefficients/Rates that need to be determined are substrate uptake, PGA production and growth.

Katja used nitrogen starved conditions to test for PHA production to limit growth and direct as much carbon to PHA as possible. For PGA this needs to be altered because PGA needs Nitrogen. This Nitrogen provision while decoupling growth and PGA production will be a novel contribution to knowledge.

Measure: substrate (e.g. acetate (for us, glycerol and citrate?) biomass, PGA (or easily analyzable proxy), CO2, O2 and N-source ammonia.

Katja’s Chapter 2: “Both SBR and fed-batch reactors were highly dynamic systems with changing reaction rates and liquid volumes, making the evaluation of experimental data a complex task. A very detailed data analysis was carried out for each SBR cycle measurement and fed-batch experiment. The data analysis included for example the correction of measurements for sampling effects and liquid volume changes, the computation of oxygen consumption and carbon dioxide evolution, and the calculation of the best estimates for all reaction rates and total conversions at each point with the help of a metabolic model.”

Different operational conditions: low sludge residence times, nitrogen versus carbon limitation (Chapter 3). Reactor temperature (Chapter 4).

Other things to consider:
PGA product recovery and impact on reactor robustness (aka the efect of harvest, what constitutes overharvest)
Phosphate. Is there a link between PGA accumulation and P-storage? (probably not investigated in this project)

uhm. next steps, working on chapter 2 – the metabolic model. I start with Katja’s work, build in the NH4 uptake and  role in PGA production and consumption, supported by the work done by the groups of Margaritis and Ashiuchi. Then start generating data to support this, with both Agnes and Roberta.