[1]

Valorization of spent anaerobic digester effluents through

production of platform chemicals using Cl. butyricum
Myrto-Panagiota Zacharof* a, b, c Clotilde Vouzelaud*a Stephen J. Mandale a, b, c
and Robert W. Lovitt a, b, c
a

Centre for Complex Fluid Processing (CCFP), College of Engineering, Swansea University, Talbot

building, Swansea, SA2 8PP, UK

b

Centre for Water Advanced Technologies and Environmental Research (CWATER), College of

Engineering, Talbot building, Swansea University, Swansea, SA2 8PP, UK

c

Systems and Process Engineering Centre (SPEC), College of Engineering, Swansea University, SA2

8PP, UK

________________________________________________________________
Abstract
Spent digester streams were reformulated into nutrient rich effluents, capable of supporting
biotechnological production of platform chemicals. This was achieved, using a set of physical pretreatments including, sedimentation, dilution and sieving, followed by cross-flow microfiltration
processing to give particle and cell free nutrient streams with a C:N:P molar ratio of 1:1.35:0.03 .
These streams were inoculated on bench scale, with Cl. butyricum NCIMB 7432, a well-known acids
producer, giving good growth rates (max 0.24 h-1) and acids concentrations of 110 mM acetic acid and
18 mM per liter butyric acid. When the treated effluent was fortified with 2 % w/v glucose there was a
significant improvement with a 0.38 h -1 max and platform chemicals concentration of 279 mM acetic
acid and 32 mM butyric acid per liter. These media gave comparable performance with the synthetic
media, therefore demonstrating a valid alternative solution to commercial media preparations. The
cost of treating the excess digestate by microfiltration within the scope of formulation to nutrient
media has been calculated at 0.0033 $USD/ kg, 3 times lower than the cost of production of nutrient
media.

Keywords: acetic acid; butyric acid; effluents; microfiltration; fermentation; valorization

[2]

Graphical Abstract

Highlights

Cl.butyricum was grown on spent digester effluents of C:N:P ratio 1:1.35:0.03

The cost of digestates formulation to nutrient media was at 0.003 $USD/ kg.

Concentration of acids reached 110 mM acetic acid and 18 mM butyric acid per liter.

Addition of 2 % w/v glucose almost doubled the acids concentration.

These media gave comparable performance with the synthetic commercial media.

[3]

1.

Introduction

The rapid increases of human population, the heavy urbanisation of the land and extensive
industrialisation of goods production, have led to the continuous intensification of waste discharge to
the environment [1, 2]. Wastewater, domestic, municipal, or industrial is a serious environmental
constraint, contributing to the phenomena of environmental toxicity and human health pathogenicity,
if discharged untreated to environment [1, 2].
Wastewater is often found to contain hazardous chemicals including metals (e.g. As, Pb, Cr, Cd and
Zn), toxic compounds such as endocrine disruptors, dyes and strong, pungent odours due to high
content of organic matter. However waste streams, have been also identified as a source of valuable
commodities including phosphate, ammonia, metal ions and volatile fatty acids (VFA) [3].
Various methods have been proposed for treating wastewater to make it safe for discharge to the
environment that require costly plant processing using extensive amounts of energy, biological
processes and additional physical and chemical treatments [4]. These treatments generally do not
allow either the recovery or the reuse of chemicals leading to the loss and dilution of important
resources [5].
On the other hand, making judicious use of pre-treatments where necessary, membrane processes can
be successfully applied to ascribe value to wastewater by recovering valuable nutrients. These
nutrients could then be precisely reformulated as nutrient growth media for microbial production of
platform chemicals. This approach could greatly benefit current industry, promoting sustainability
since the wastes are not released untreated to the environment causing phenomena of soil toxicity,
eutrophication and microbial contamination [6].
Recovery and separation of valuable nutrients from wastewater with its diverse composition and
complex physicochemical nature calls for innovative cost-effective engineering methods and
strategies for recovery of materials. Within this context, low energy physical treatments such as
dilution, sedimentation and filtration/diafiltration using pressure driven membrane technology can
offer a viable solution. Membrane technology is still a rapidly developing technology and offers a cost
effective option, as it is easily scalable with numerous arrangements and alternatives and the
technology is often easy to incorporate and integrate into waste treatment processes. They offer high
productivity and low operational cost compared to other competing technologies since there is no
phase change required and minimal or no use of chemical additives [7]. Using this technology, waste
can be recycled back to the production systems substituting for newly manufactured materials.
Particle separation can be achieved with a wide range of membranes technologies covering
microfiltration, ultrafiltration, nanofiltration and reverse osmosis while the substances of interest can

[4]

be clarified, fractionated, and concentrated to produce high value streams at low cost [8].
Using this technology wastewater itself can be valorised (Fig.1.) by removing coarse particles,
indigenous microbial/viral load, toxic substances and colorants. These materials once separated and
concentrated into streams of nutrients, organics and salts can then be precisely formulated to serve as
growth media for microbial production of platform chemicals and biofuels.
These materials, if used as nutrient media, are potentially highly profitable, especially when compared
to the traditional synthetic media or those derived from food sources such as crops. Filtration allows
manipulation of the nutrient content, since it can be combined with leaching and acidification using
MF or selective separation and concentration using subsequent NF and RO processes. These streams
can then be blended enabling the formulation of different concentrations of appropriate proportions
[9] suitable for supplying the nutritional needs of microbial fermentations for the intensive production
of biofuels, acids and other chemicals such as lipids and enzymes [10].
For example, for the production of acids by Clostridia spp., the commonly used complex synthetic
medium has high (30 g/L) nitrogen content due to ammonium salts, yeast extract and peptone. If
waste effluents were used instead, the nitrogen sources could be supplemented via organic content in
the form of ammonia and the other components such as phosphate and metals.
Clostiridia spp., and especially Cl.butyricum is of great interest to the industry (pharmaceutical, food
and environmental) as a natural- acid producing- bioreactor. Cl. butyricum, commonly cultured in
mesophilic, neutral to alkali, microaerophilic conditions [11] produces a mixture of acetic and butyric
acid simultaneously with hydrogen and carbon dioxide.

Acetic acid has a global size market of

3.500.000 tonnes per year with a price range of 400-800 US dollars ($) per tonne while at about
50.000 tonnes of butyric acid are produced per year with a market price ranging between $2000-2500
a tonne [12].
Of the global demand for acids, only a percentage is covered by recycling, the majority is produced
from petrochemical feedstock [5]. However, driven largely by legislative, environmental, economic
and social drivers, focusing on uncoupling the generation of energy and chemicals from fossil fuels in
an effort to decelerate the climate change, the industry is shifting towards the production of platform
chemicals from alternative sources,[13,14] using environmentally friendly methodologies including
fermentation.
Consequently, envisaging waste as a virtually inexhaustible mine is an important step to the
development and deployment of alternative sources for energy production, achieving environmental
sustainability and constituting waste safe for environmental discharge in the form of particle free,
nutrient free and sterile effluents.

[5]

Therefore this work reports on the use of membrane microfiltration for the formulation of spent
digester effluents into nutrient media, suitable for microbial growth of industrially important
microorganisms. The prepared microfiltered effluents were then physicochemically characterized
before being assessed for their utilization as growth substrates of microorganisms associated with
chemical and fuels production. As an example, these formulated media were then used to assess the
performance of Cl.butyricum, in bench scale batch anaerobic cultures. Comparative studies were done
using synthetic growth media, minimised media and enriched anaerobically digested filtrates. These
studies were then used to perform, a comparative cost estimation study to assess the potential
economic impact of this proposed methodology.

2. Materials and Methods

2.1. Materials
2.1.1. Chemicals
Yeast extract, glucose, magnesium sulphate, potassium dihydrogen phosphate, and sodium hydroxide
(NaOH) were obtained from Sigma-Aldrich (Gillingham, UK).

2.1.2. Inoculum source

Cl. butyricum NCIMB 7423 was provided in a lyophilised form by National Collection of Industrial
Food and Marine bacteria (NCIMB), Aberdeen, Scotland, United Kingdom. The bacterium was
revived twice by inoculating the selected strain into 50 ml serum vials containing optimised liquid
medium (yeast extract 10 g/L, glucose 10 g/L, ammonium sulphate 5 g/L and 2.5 g/L potassium dihydrogen orthophosphate ) and were statically incubated at 37C (Thermo Scientific Series 6000
Incubator, USA) for 24 hours. Stock culture solutions of each strain were made through
cryopreservation method. For constant use, the bacterium was regularly inoculated (on a weekly
basis) into 30 ml serum vials containing basal medium and were preserved at 2 C [15].

2.1.3. Waste Effluents

Waste effluent streams (agricultural wastewater derived from spent agricultural digested sludge
namely mixed waste of cattle slurry, vegetable waste and silage), taken off the output line of the
anaerobic digester (AD) used for manure production but before passing through the automatic coarse
particle separator (>5mm), were collected off Farm Renewable Environmental Energy Limited (Fre),
Wrexham, United Kingdom (http://www.fre-energy.co.uk/). These samples were pre-treated using
dilution, mixing, sedimentation and sieving [8] in a 150 L capacity stainless steel vessel. The resulting

[6]

effluents were microfiltered through a pilot scale unit equipped with a ceramic membrane (pore size
<0.2m) [8]. The resulting particle free, sterile effluents were used as nutrient media in this study.

2.2. Methods
2.2.2. Filtration Unit Design
A cross-flow microfiltration unit was developed equipped with a ceramic membrane (Fig.2) to
process the waste. The unit comprised a 100 L volume capacity stainless steel cylindrical feed tank
linked through 5 m of 1 inch stainless steel pipes, arranged into two fluid loops each one driven by a
centrifugal pump type Kennet 12-2 (Stuart Turner, UK). AD Waste was circulated from the tank into
the first pump loop which pressurised the system against a diaphragm valve (Axium Process, Hendy,
Wales, UK) on the return side, which could be adjusted to control the pressure applied to the system.
Within this loop an additional pump was used to feed at high flow rates the membrane and water
cooled heat exchanger in series. The membrane used here was a Membralox ceramic (-Al2O3)
monolith microfiltration module (pore size 0.2m), able to withstand a pH range between 0-14. This
membrane was fitted in stainless steel housing, commercially available by Pall (Portsmouth, UK).
This arrangement allowed limited pressure drop in this loop therefore high fluid velocity over the
membrane surface was achieved, which could be kept constant over a range pressures. The membrane
comprised 19 channels, of 3.70 mm diameter each and length of 1016 mm. The effective membrane
area was determined as 0.22m. All the parts of the unit were connected with stainless steel hygienic
clamped flanges with PTFE seals, provided by Axium Process, (Hendy, Wales, UK).

2.2.1. Growth on optimised liquid media and minimised media

The specified quantities of powdered materials for optimised liquid medium, namely, yeast extract 10
g/L, glucose 10 g/L, ammonium sulphate 5 g/L and 2.5 g/L potassium di-hydrogen orthophosphate,
were weighed on an electronic balance (Sartorius, CP4202S, JENCONS-PLS, Germany) and they
were added into an Erlenmeyer flask containing 1L of distilled water. Once mixed the medium (pH
5.01) was decanted under gaseous nitrogen flow, achieving microaerophilic conditions, into 250 ml
Erlenmeyer flasks in 100 ml aliquots. The flasks were sealed and secured with cotton and aluminium
foils and were autoclaved at 121 C for 15 min and left to cool overnight. The same process was
followed for the preparation of minimised media (tap water mixed with 2% w/v glucose in a 1:1
ratio). The flasks were gently mixed using a vortex mixer and inoculated with 10 ml of inoculum.
They were then incubated in a rotary shaker at 37 C (Thermo Scientific Incubator, UK) [16].

[7]

2.2.2. Growth on treated agricultural wastewater and enriched agricultural

wastewater
The treated agricultural wastewater was decanted under gaseous nitrogen flow, achieving
microaerophilic conditions, into 250 ml Erlenmeyer flasks in 100 ml aliquots. The medium having
passed through a microfiltration membrane was considered sterile. The treated agricultural wastewater
used as nutrient medium was then enriched with 2% w/v (glucose solution added in a 1:1 ratio).

2.2.2. Measurement of cellular growth and biomass

The cellular growth was measured into a UVVisible UNICAM UV300 dual beam spectrophotometer
at 600 nm. The tube had a 1 cm. light path. Maximum specific growth rates (max, h-1) of the microbial
strain were determined in a 10 hour cycle of incubation at 37C in a rotary shaker at 50 rpm allowing
minimum gas exchange (Thermo Scientific Series 6000 Incubator, USA). To convert optical density
(OD) measurements into dry weight units (g L-1) of bacteria, dry weight determination assays were
performed [15], resulting in a linear equation (two variables) with an intercept-slope of the form
y=mX+b for dry weight determination where X stands for OD units. The equation for Cl.butyricum
was Y=0.0959 X +0.0006 [17].

2.2.3. Physicochemical characterisation of the treated agricultural wastewater

Total solids (TS, g/L), total suspended solids (TSS, g/L), total dissolved solids (TDS), alkalinity,
optical density, nitrogen measured as ammonia (NH3N) and phosphorous (PO4P) using the phenate
and vanadomolybdo-phosphoric acid colorimetric methods were determined according to Standard
Methods for the Examination of Water and Wastewater published by APHA, AWWA and WPCF 20th
Edition, 1998. VFA were determined using head space gas chromatography [7]; offering highly
significant results. Particle size distribution (PSD) of the sludge samples was determined by light
scattering technique using Mastersizer 2000 (Malvern, UK), the zeta potential was determined by the
Zetasizer (Malvern, UK),the conductivity and salinity of the samples were measured used a
conductivity meter (Russell systems, UK) calibrated with a standard solution of 0.1M of KCl.

2.2.3. Analysis of end products using gas chromatography

Volatile fatty acids, butyric and acetic acid were analysed utilising head space gas chromatography
(GC), VARIAN ProStar GC-3800 (USA), equipped with a Nukol, fused silica high-quality coated
polyamide capillary column 15 m x 0.32 mm I.D., 0.25 m column. The GC was connected with a

[8]

hydrogen generator (UHP-20H NITROX, Swan Hunter, UK), and an air supply. Helium was used as a
carrier gas. Analysis was conducted according to the following protocol: of a total holding time of 15
minutes, a gas flow rate of 30 ml/min and a pressure of 10 psi and an FID temperature of 220 C as
described by Sigma-Aldrich GC Supelco-Nukol columns manual.

2.2.4. Numerical Analysis of the Experimental Data

Each differential parameter was triplicated to obtain the average data. The data were statistically
analysed for accuracy and precision calculating standard deviation, standard error, experimental error,
regression factor and reading error (Microsoft Excel software Version 2007). All the numerical data
were proven to be highly accurate and reproducible having a mean standard deviation of below 5%
and experimental error below 5% offering highly significant results.

2.3. Cost Estimation

2.3.1. Process Description
However, the wide adoption of such a waste processing scheme is strongly influenced by the cost
efficiency of this application when compared to either the conventional methods of waste treatment or
production of defined and semi defined commercially available nutrient media. Estimating the cost of
these processes though is rather complicated as several factors have to be taken into careful
consideration, such as capital cost related to manufacturing and maintenance of the system and
relevant equipment, labour costs, energy consumption and transportation of waste.
To investigate the feasibility of using agricultural waste as nutrient media for industrially relevant
fermentations, a costing study was conducted using the factorial method of cost estimation [30-32].
The study was based on the development of a decentralised, (treatment on local site) microfiltration
unit. The unit would be able to treat 10 m3/h of excess pre-treated agricultural waste. The cost of the
produced effluents of such a system was compared with formulation of commercially available
synthetic nutrient media. All costs are given in 2014 USD $. Where necessary costs were converted
using the Marshall Swift Index (MSI) for equipment, the Producers Price Index (PPI) and the
Consumer Price Index (CPI) for miscellaneous costs [30-32]. The location of both units is assumed to
be in the United Kingdom.

2.3.2. Design and Cost of the Units

The basis of the analysis is the treatment on a daily basis of 220 m3 of agricultural sludge and 220 m3
per day of formulated nutrient broth made of powdered materials and deionised water. Since the

[9]

waste-to-nutrient media membrane system was designed to treat 10 m3/h, 2 hours per day are assigned
to cleaning and maintenance of the units. Both units are made of stainless steel 304 with dairy fittings.
The waste treatment unit (Fig.4) is attached to a conical-base flat-roof settling tank to which the
excess agricultural sludge is pumped from the digester. The membrane selected is a microfiltration
ceramic alumina zirconia membrane of 107 monolith modules each one composed of 19 channels [37,
38]. All components of the unit are commercially available from numerous companies in the United
Kingdom and worldwide. The media preparation unit (Fig.5.) is designed as a cylindrical vertical flat
base vessel with a mixer for homogenous mixing and a flat top, with an input valve .The unit is
equipped with pressure gauges, pH and temperature meters and level gauges with the equipment used
being commercially available.

2.3.3. General Economic Parameters

Operating costs can be broken into several main categories including equipment, labour, maintenance,
utilities and raw materials (Table.3). The total investment cost (TIC, $) is calculated by adding fixed
capital (FC, $) and working capital (WC, $) [30, 31]
TIC= FC+WC
The direct production costs (DPC, $) or annual operating cost (AOC, $) are calculated by adding
variable costs (VC, $) and fixed costs (WC, $)
DPC=AOC= VC+WC
The production cost (PC, $/kg) is calculated by annual operating cost (AOC, $/year) divided by the
annual production rate (APR, kg/year).

PC ( / kg)

AOC,$ / year
APR , kg / year

3. Results and Discussion

3.1. Physicochemical Characteristics of Agricultural Waste Effluent Streams
Twenty-five liter (25 L) sludge samples were taken from the anaerobic digester without any on site
processing. These materials were found to be rich in particulates such as coarse particles, stones and
straw. A pretreatment scheme combining dilution, thorough mixing, sedimentation, and sieving was
devised, aiming to remove large particulates (>1000 m) and facilitate the effluents' filterability
through the microfiltration unit. Dilution allowed the disengagement of the chemicals and nutrients of

[10]

interest namely ammonia, volatile fatty acids, phosphorus and metal ions from the solids. On the other
hand sedimentation ensured the settling of heavier particles out of the fluid and their resting towards
the bottom of the sedimentation tank, while sieving of the liquid phase through a series of coarse
filters (> 500 m) resulted in the removal of any large suspended material such as smaller pieces of
straw or sand. In parallel to the successful removal of large solid matter, it was also possible to
recover important nutrients in the supernatant fluid that are normally loosely associated with the
solids. TS and TSS content was reduced by 20.75% and 58.75% correspondingly, while the mean
particle size dropped by 48.58%, while there is a partial loss of nutrients. Of all the chemicals of
interest, phosphate was mostly affected showing a 34.05% reduction while the content of metal ions
was reduced by 21.32% (Table 1).
The pre-treated effluent was found to be rich in substances of high nutritive value, suitable for
microbial fermentations. However, the high solids and organic matter content were preventing the use
of the pre-treated effluents in their current form, mostly due to potential difficulties in the recovery of
the end products of the microbial metabolism. Therefore the effluents were further treated using
membrane technology, namely microfiltration, providing an effluent in a condition suitable to be used
as nutrient source for microbial fermentations and thence production of biofuels and chemicals.
Microfiltration can be used to effectively retain solids and organic residues i.e. color, allowing the
passage to the permeate of the nutrients of interest. This resulted in the formation of a nutrient rich
sterile and particle free solution.

3.2. Physicochemical Characteristics of Treated Agricultural Waste Effluent

Streams
The behavior of the unit was analyzed by measuring the permeability of tap water in varying pressure
conditions (increase in outlet pressure 0 to 20 psi). The flux and cross flow velocity values were
increased linearly with increasing pressure., ranging from 148 to 539 L/m h and the cross flow
velocity increased from 3.05 m/s to 10.89 m/s. The membrane permeability (L) characteristic of the
unfouled membrane, calculated as 18.5 m, was defined by the slope of the linear functions using the
plots of the flux over the transmembrane pressure (TMP). The system is designed and developed to
operate efficiently at various pressure conditions, allowing high productivity. Since high flux and
cross flow velocity can be achieved at low pressure conditions, it was decided to process the pretreated wastewater at low pressure conditions to enable the development of a cost effective, due to the
controlled energy consumption, scalable mechanical treatment of agricultural wastewater, in the
context of recovery of valuable nutrients.
The coarse, particle free, wastewater effluent was filtered through the ceramic cross-flow
microfiltration unit at a TMP of 15 psi, achieving a flux of 103.32 L/ m2 h. During filtration, the

[11]

majority of solids and insoluble organic matter was retained by the membrane filter (Table 1).
Interestingly, the cross flow arrangement of the filtration unit allowed the continuous circulation of
the processing fluid in the system. This enabled the continuous disengagement of nutrients retained in
the compressible permeable cake formed by the deposition of solids in the membrane channels. These
were transferred to the permeate allowing the formulation of a complex but particle free solution to a
molar C: N: P ratio of 1:1.35:0.03. These components can successfully be used by microorganisms as
growth stimulants (nitrogen, carbon and hydrogen intake). However, the solid matter content, the
conductivity and the ions related indicate a solution rich in mineral salts that may be uptaken during
the microbial metabolism. These may hinder intensive growth of the propagated microorganisms or to
microorganisms whose end products are susceptible to genetic mutations such as enzymes or proteins
that might be proven to be toxic for the end products. For bacteria or fungi though with metabolic
products such as acids or biofuels like ethanol these effluents can be used safely.

3.3. Assessment of Treated Agricultural Waste Effluents as Nutrient Media

Cl. butyricum is a well-known mixed acids producer, with numerous applications in industry,
including the pharmaceutical, food and environmental sectors. Cl. butyricum has been identified as
part of the microbial group participating in anaerobic digestion process as part of the acetogenesis
phase, producing acids and hydrogen [18]. Butyric and acetic acid, which are the main end products
of its fermentative metabolism, are used as food flavour enhancers and anticontaminant agents with a
substantial market size per year [12].
Aside from the end products of its metabolism, Cl. butyricum adaptability to varying physicochemical
conditions including aeration, pH, temperature and its ability to metabolize a wide range of
carbohydrates and other sources made this strain an ideal candidate for biotechnological production of
acids using waste. The formulated waste effluents (Table 1) were then used to assess the performance
of Cl. butyricum NCIMB 7423, in bench scale batch anaerobic cultures. Comparative studies were
conducted among the formulated waste based media; minimized water based media and standardized
optimized in vitro media.
When compared to synthetic growth media, good growth (Fig. 3) was achieved with the
microorganism using filtrates; however there was a significant reduction of growth rate, 51% and in
the total acetic acid and butyric acid produced of a 61%. The treated effluents supported the growth of
the bacterium sufficiently producing in total 118.8 mmols/ L of acids (Table 2) however the synthetic
in vitro media offered a higher amount of acids and biomass. The microorganism was able to grow
but not produce any acids, in minimized media, where a reduction of growth rate of 71.4 % occurred.
The minimized media in other words sterile tap water enriched with glucose proving the strong impact
of carbohydrate on the microorganisms growth and acids production.

[12]

Therefore, when the treated effluents were supplied with 2% w/v glucose solution a significant
improvement in the growth rates and growth yields, 36.84% and 28.67 % respectively were seen
when compared to the treated digested agricultural wastewater. The addition of glucose boosted the
production of acetic acid, achieving a higher amount than synthetic media while the highest rate of
butyric acid was achieved in the synthetic media. Regardless of the acetic acid (3.500.000 tonnes
/year) and butyric acid (50.000 tonnes/year) market size [5,45], limited studies have been conducted
regarding their biotechnological production on waste streams, and to the authors present knowledge
none on anaerobically digested effluents. Several researchers [46-49] employing extensively
pretreated (enzyme, acid hydrolysis, maceration) carbohydrate rich wastes such as sugarcane bagasse,
waste paper, apple pomace and cheese whey, have achieved a maximum acetic and butyric acid
concentration of up to 667 mmols/L and 567.47 mmols/L. Apart of the nutrient rich extensively
pretreated waste effluents used, these studies have been optimizing the culturing methodology
employing large scale continuous culturing techniques improving significantly the yield of acetic and
butyric acid. However, these studies have not been evaluated in terms of cost effectiveness and
operation while this type of waste has several other competitive uses as biotechnological feedstock
such as their use in the production of polyhydroxyalkonoates (PHA, PHB, PLA).
Powdered glucose however is an expensive additive, with a current bulk value of USD $400/ton. It
could be replaced by alternative sources, such as dairy whey that has been estimated to contain 48 g/L
of lactose and 10 g/L of protein [19] or confectionary and sugar processing waste such as corn syrup
or molasses. Using dairy whey as a supplement would be advantageous, since it has a high nutrient
content and already requires extensive treatment before disposal. From the annual production of whey
in Europe about 13.4 million tons remains as surplus product, and its processing and treatment is
expensive especially for small and medium cheese producing industries [20]. Whey has been proven
to be readily utilized by numerous microorganisms including clostiridia spp. [21] enhancing the
productivity of acids and ethanol. Consequently whey is an attractive option to replace glucose
powder and achieve high acids concentrations.
A further benefit of this approach, ergo the use of treated waste effluents as nutrient media, is the
minimization of the use of yeast extract. Yeast extract is a protein and nitrogen rich, up to 85%
composition, microbial growth supplement. It is traditionally produced by virgin yeast cells grown on
beet or cane molasses on a batch or fed batch mode [22, 23]. Although containing carbohydrate
sources up to a 75%, molasses do not fully support yeast growth, therefore vitamins (biotin, thiamine,
pantothenic acid) and magnesium and potassium salts are added [24]. The high cost of raw materials
as well as the sophisticated techniques used for the downstream processing (membrane filtration,
spray drying) have elevated the cost of yeast extract up to $3000 USD/ton [24]. Substituting the use of
yeast extract with a nitrogen rich solution derived from waste would be highly profitable as well as

[13]

beneficial for the environment since yeast industry wastewater, is characterised by high chemical
oxygen demand (COD) often above 25,000 mg/L, dark colour, and high concentrations of total
nitrogen and non-biodegradable organic pollutants, demanding extensive treatment prior to discharge
[25, 26].

3.4. Annual Cost Production

The application of this technology will depend on its practical and cost effective application to the
formulation of nutrient media, tailored in compositions to the nutritional needs of the bacteria of
interest. Estimation of costs is complex as they arise from a variety of factors, such as energy
consumption, addition of water, labour and maintenance and capital costs, such as equipment and
scale of operations [33-36]. The value of the products is dependent on the concentration of nutrients
present and this may be compared to the costs of equivalent materials such as in vitro nutrient media.
The cost of treating the excess digestate by microfiltration in order to formulate it into nutrient media
in other words the production cost was calculated as $USD/ kg of treated effluents, as 0.0033 $USD/
kg. On the other hand, the cost of preparation and production of standardised nutrient media was
found as 0.0094 $USD/ kg respectively. Therefore the cost of production of sterilised nutrient media
is almost 3 times higher than the cost of waste treatment.
Although of the cost of mechanical equipment required for the waste treatment unit is higher than the
nutrient media preparation system (Table 3), the operational costs are low (Table 4). Since the unit is
equipped with a membrane filter, sterilisation is achieved through filtration rather than continuous
steaming of the mixture, therefore the cost in utilities is low. The use of electricity is lower in the
waste treatment system as the system has been designed to operate with two pumps using the cross
flow arrangement which supports mixing, while the nutrient media system is equipped with six
propellers to achieve continuous homogenous mixing. Furthermore, the water needed for nutrient
media formulation is not used in the waste treatment system and the cost of excess agricultural
wastewater as raw material is a nonentity.
On the other hand, the cost of powdered chemicals for the nutrient media is quite high [39-41]. The
calculations are based on 2014 price catalogues provided by nutrient media distributors and
manufacturers. It is evident, that the use of waste as nutrient source is effective and economical as
well as environmentally advantageous, since the production of powdered yeast extract has a carbon
footprint of 0.936 kg CO2 per kg of material [43].
The additional cost of the synthetic media steam based sterilisation has been calculated as $USD
141,209.57 per year. However, sterilisation via steam can be replaced by microfiltration [44], where
the cost would be equivalent $USD 56,658 per year, based on the use of filtration equipment in this

[14]

study. These costs have not been included in the calculations, since in the industry sterilisation would
normally occur in situ, in the bioreactor; therefore the media can be provided unsterilized.
The microfiltered effluents have been successfully valorized since coarse particles have been
removed, as has the indigenous microbial load, toxic substances and colorants. Filtration also allows
manipulation of the nutrient content, since it can be combined with leaching and acidification using
microfiltration or selective separation and concentration using subsequent nanofiltration and reverse
osmosis processes. This approach has several advantages such as: recycled materials that will
substitute for newly synthesized or mined materials; the reduction in the volume and concentration of
waste will reduce demand and costs in waste treatment plants; and the creation of valuable streams
such as those formulated from nutrient streams for application in agriculture and bioprocessing.
A medium size anaerobic digester is able to treat 11000 -15000 m3 of organic waste (cattle manure,
chicken manure, vegetative waste) per year within the scope of manure production and biogas
generation [27]. Regardless of the effectiveness of the process, AD is dependent on various factors
including the feed composition, the hydraulic retention time and the environmental conditions
(weather, socioencomic factors), and as such an amount of excess digestate is generated [26]. The
excess of untreated material forms a level of grit in the digester that gradually blocks the digester
function, and since the mixing cannot be homogenous this can result in a change to the
physicochemical conditions of the process such as water activity. This does alter the natural microbial
flora performing the multistage anaerobic digestion process. This affects the output, such as manure
quantity and quality as well as biogas quantity and composition [28, 29]. Therefore removing the
excess sludge ensures the continuous function of the digester, benefiting financially the industry by
avoiding the disruption of digesters function due to cleaning. Excess sludge could be removed by
pumping to a locally sited pre-treatment tank and then to a feed tank connected to a microfiltration
unit.
Untreated disposal of animal waste can cause health hazards related to microbial load as well as toxic
compounds that can be potentially dangerous to human health. The application of simple physical and
mechanical treatment, including sedimentation and followed by microfiltration offers an effective
alternative to the traditional methodologies for waste management. It can possibly facilitate the
formulation of microbial particle free effluents, safe for discharge into the environment.
This approach can certainly benefit industry at a regional and national level through the use of a
relatively abundant inexpensive feedstock that is able to be recycled to produce high value chemicals
while reducing the carbon footprint of fermentation and reducing waste disposal. Such a system,
namely the development of a complete membrane processing strategy within the scope of nutrient
recovery, can be effectively integrated in to the existing systems of waste treatment, for example in

[15]

wastewater treatment plants or in small, medium and large enterprises incorporating anaerobic
digesters for treatment of waste. These effluents, if utilized as nutrient media, are potentially highly
profitable, especially when compared to the traditional synthetic media or that derived from food
sources such as crops.

4. Conclusions
It can be concluded from the experimental results presented here that treated agricultural wastewater,
can successfully support the growth of Cl. butyricum. The formulated effluent is suitable for use in
large quantities due to its low cost of formulation and its content of nutrient sources. The effluent can
support the production of platform chemicals at satisfactory levels. When the treated effluents are
enriched with glucose the platform chemicals production is enhanced and reaches comparable levels
to the production of the in vitro media.

Successful valorization of the waste effluents has occurred.

The cost of treating the excess digestate by microfiltration within the scope of formulation to
nutrient media has been calculated at 0.0033 $USD/ kg, 3 times lower than the cost of
production of nutrient media.

The wide adoption of this methodology will depend on the its practical and cost effective
application

Acknowledgements
This project was supported by Low Carbon Research Institute (LCRI) project grant title Wales H2
Cymru. The authors would like to thank Mr. Chris Morris, Technical Director and Ms. Denise
Nicholls, Business Manager, Fre-energy Farm, Wrexham, Wales, United Kingdom, for providing the
team with anaerobically digested agricultural wastewater.

[16]

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[37]

TAMI

Industries

INSIDE

CeRAM

http://www.tami-industries.com/INSIDE-CeRAM-

TM.109+M54a708de802.0.html (lastly assessed 19th December 2014)

[38] Pall Corporation Membralox Ceramic Membrane Productshttp://www.pall.com/main/food-andbeverage/product.page?id=41052 (lastly assessed 19th December 2014)
[39] Melford Chemical and Biochemical Manufacturing price catalogue http://melford.co.uk/(lastly
assessed 19th December 2014)
[40] LabM Limited price catalogue http://www.labm.com/(lastly assessed 19th December 2014)
[41] Neogen Corporation, Acumedia Manufacturing http://www.neogen.com/Acumedia/ (lastly
assessed 19th December 2014)

[19]

[42]

Collister

&

Glover,

Lowara

SHE

Centrifugal

Pumps

https://www.colglo.co.uk/product.php?product=LOWAP107410020 (lastly assessed 19th December

2014)
[43] Pia Karjalainen, P., Bin The carbon footprint of the Finnish beverage industry for years 20002012 as calculated with CCaLC. Master's Thesis, Helsinki: University of Helsinki; 2013.
[44] Monteil et al.: The optimization of a rapid low-cost alternative of large-scale medium
sterilization. BMC Proceedings 2013 7 (Suppl 6):P45.
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of advanced biorefinery schemes to be integrated into existing industrial fuel producing complexes
project report [ONLINE] Available at: www.bioref-integ.eu. [Accessed 14 April 15]
[46] Kaur I, Ni Yi. A process to produce furfural and acetic acid from pre-hydrolysis liquor of kraft
based dissolving pulp process. Sep. Pur. Tech. 2015; 146:121-6
[47] Parmar I, Vasantha Rupasinghe, HP. Bio-conversion of apple pomace into ethanol and acetic
acid: Enzymatic hydrolysis and fermentation. Biores. Tech. 2013; 130:613-20
[48] Nayak J., Pal M., Pal P. Modelling and simulation of direct production of acetic acid from
cheese whey in a multi-stage membrane-integrated bioreactor Biochem Eng. J 2015; 93:179-95
[49] Jiang L, Wang J. Liang S, Wang X, Cen P. Xu Z. 2009. Butyric acid fermentation in a fibrous
bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses. Bioresour. Technol.
2009, 100:3403 9.

[1]

Parameters

Total Solids (TS, g/L)

Total Suspended Solids (TSS,
mg/L)
Total Dissolved Solids (TDS, mg/L)
Conductivity (mS/cm)
Optical Density (580 nm)1
pH
Zeta potential (mV)
Sizing (m)
Concentration
Acetic Acid
Butyric Acid
Metal ions (Ca,Cu, Co,Fe, Pb, Mg,
Mn, Zn,K, As)
Ammonia
Phosphate
Alkalinity (mg CaCO3/L)

Untreated
digested
agricultural
wastewater
15.13
612.50

Pretreated digested
agricultural wastewater

Treated digested agricultural

wastewater microfiltered (0.2
m) retentate

Treated digested agricultural

wastewater microfiltered (0.2
m) permeate used as growth
media
6.04
190.00

11.99
252.60

10.40
258.00

7964
9.37
0.86
8.37
-33.25
27.17

7743
9.11
0.34
8.11
-30.06
13.97

4250
5.30
0.10
8.25
-24.2
2.93

1650.17
1781.58
1462.86

1464.02
1666.16
1150.91

7657.95
9.01
0.27
8.01
-29.60
13.49
mg/L
1083.30
1163.93
769.41

1577.08
624.91
8750

1494.54
412.10
7500

958.79
351.62
5050

686.19
41.51
2287

1265.85
1393.02
880.00

Table 1: Physical characteristics and chemical composition of the untreated and pretreated anaerobically digested agricultural sludge

The collected samples were diluted 100 times with deionised water and measured in a 1 cm light path

[2]

Growth media

Growth rate (max, h-1)

Final Biomass
Concentration (g/L)

Platform chemicals concentrations

0.49

1.92

257.1

Butyric acid
(mmols/L)
73.9

0.14

0.88

0.24

1.36

110.9

17.9

0.38

1.75

279.4

31.9

Bacterial strain

Cl. butyricum

Invitro standardised
growth media
Minimised Growth
media
Treated digested
agricultural
wastewater
Enriched Treated
digested agricultural
wastewater

Acetic acid (mmols/L)

Table 2: Comparison of the effect on Cl. butyricum growth and platform chemicals production on waste based media and synthetic media

[3]

Units

Element

Cross flow
Tanks
microfiltration
unit
(waste
Pumps
treatment)
Membrane
Heat
exchanger
Raw
Materials
Nutrient broth Tank
preparation
unit
Propeller
and baffles
Raw
Materials

Type

Settling
Processing
Collection
Feed
Recirculation
Monolith tubular
Shell and tube

Surface
area
(m2)
319.83
201.56
201.56
3.46

Spent digester
effluents
Processing

Mixing

59.72

Powdered
chemicals i.e.
yeast extract,
glucose, sodium
chloride etc. and
tap water

201.56

Material

Total Cost
(USD, $)

Stainless steel
Type 304

77,799.50
75,593.64
75,593.64
8,606.252
8,606.25
26,864.553
10,555.98

Plastic/Metal
Ceramic
Stainless steel

Stainless steel
Type 304
Stainless steel
Type 304
Powder or
liquid

Power
Usage
(MJ)
-

Steam
Usage
(kg/d)

Cooling
water
(kg/d)

286.12
1800

220000

1800

75,593.64

25,195.96

103.10

519,729.664

Table 3: Major equipment specification and purchase cost (based on 2014) to obtain 220 m3/d of media

Price is based on Lowara She 32-125/07/a pump 3 phase centrifugal 0.75kW (Lowara pumps, UK). Its specification are maximum delivery up
to 18 m3/h , Motor :400v 3 ph 50 HZ 0.75 kW at 12 bar[42]
3
Price is based on microfiltration ceramic tubular membrane of a (0.2 m pore size) by TAMI Industries (France)[37]
4
Prices are based to Melford Chemical and Biochemical Manufacturing price catalogue http://melford.co.uk/ 2014, LabM Limited price
catalogue []http://www.labm.com/ 2014, Neogen Corporation, Acumedia Manufacturing http://www.neogen.com/Acumedia/ 2014 [39-41]

[4]

Fixed capital estimate summary

Total plant direct cost (TPDC) (physical cost)
Equipment erection
Piping
Instrumentation
Electricals
Buildings
Utilities
Storage
Site development
Ancillary buildings
Design and Engineering
Variable Costs
Raw materials
Miscellaneous materials
Utilities Cost
Steam
Cooling water
Power
Water
Shipping & Packaging
Fixed Costs
Maintenance
Operating labour
Plant overheads
Capital charges
Insurance
Local taxes
Royalties
Sales expenses
General overheads
R&D
Total annual production rate(rounded)

Waste to media conversion unit (cross flow microfiltration unit

)

Synthetic media preparation unit

0.4
0.7
0.2
0.1
none required
not applicable
provided in PCE
not applicable
none required
0.3

$271,398.31

0
0

$519,729.66
$1,114.19

$16,289.57
$6,787.32
$3,884.08
$6,787.32
not applicable

$16,289.57
$1,111,37
$27,182.98

$27,794.74
$88,020.00
$44,010.00
$72,266.33
$5,558.95
not applicable
none required
not applicable
not applicable
not applicable

$11,141.91
$88,020.00
$44,010.00
$28,968.96
$2,228.38

$739,797.02

Table 4: Economic analysis results (based on 2014 prices) to obtain 220 m3/d of waste based and synthetic nutrient media

[5]

Figure 1. Schematic diagram of processing scheme of complex waste effluents and their use for intensive production
of platform chemicals

[1]

Figure 2. Schematic diagram of pilot scale filtration unit [7]: [1] feed vessel, [2] butterfly valve, [3] feed pump, [4] pressure gauge, [5] heat exchanger, [6]
pressure valve, [7] pressure gauge, [8] ceramic microfiltration unit, [9] regenerative pump, [10] drain

[2]

In vitro optimised liquid media

Minimised liquid media
Treated agricultural wastewater
Enriched treated agricultural wastewater

2.5

Biomass (g/L)

1.5

0.5

0
0

10

15

20

25

30

Time (h)
Figure 3. Growth of Cl. butyricum in vitro optimised liquid media (), minimised liquid media (), treated agricultural wastewater () and enriched treated
agricultural wastewater()

35

[3]

10

7
18
A

9
B

11
17

13
12
16

E-23

5
2

15
4

Permeate

14

Figure 4. Schematic diagram of large scale waste treatment microfiltration unit : [1] treated effluents collection vessel, [2] butterfly valve, [3] drain, [4] feed
pump, [5] pressure gauge, [6] ceramic microfiltration unit, [7] pressure gauge, [8]flow meter, [9] feed vessel ,[10] pretreatment vessel, [11] butterfly valve,
[12] standing base ,[13] prefilter unit,[14] butterfly valve ,[15] regenerative pump,[16] flow meter,[17] heat exchanger ,[18] control panel

[4]

Permeate

Figure 5. Schematic diagram of standardised nutrient media unit: [1] entrance valve [2] mixer , [3] mixing vessel, [4] paddles, [5] permeate valve