[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 pre-

treatments 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 di-

hydrogen 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

[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 m

3

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).

yearkgAPR

yearAOCkgPC

/,

/,$)/(

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 pre-

treated 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]

References

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assessed 19th December 2014)

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2009, 100:3403 9.

[1]

Parameters Untreated

digested

agricultural

wastewater

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

Total Solids (TS, g/L) 15.13 11.99 10.40 6.04

Total Suspended Solids (TSS,

mg/L)

612.50 252.60 258.00 190.00

Total Dissolved Solids (TDS, mg/L) 7964 7743 7657.95 4250

Conductivity (mS/cm) 9.37 9.11 9.01 5.30

Optical Density (580 nm)1 0.86 0.34 0.27 0.10

pH 8.37 8.11 8.01 8.25

Zeta potential (mV) -33.25 -30.06 -29.60 -24.2

Sizing (m) 27.17 13.97 13.49 2.93

Concentration mg/L

Acetic Acid 1650.17 1464.02 1083.30 1265.85

Butyric Acid 1781.58 1666.16 1163.93 1393.02

Metal ions (Ca,Cu, Co,Fe, Pb, Mg,

Mn, Zn,K, As)

1462.86 1150.91 769.41 880.00

Ammonia 1577.08 1494.54 958.79 686.19

Phosphate 624.91 412.10 351.62 41.51

Alkalinity (mg CaCO3/L) 8750 7500 5050 2287

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

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

[2]

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

Bacterial strain

Growth media Growth rate (max, h-1

) Final Biomass

Concentration (g/L)

Platform chemicals concentrations

Acetic acid (mmols/L) Butyric acid

(mmols/L)

Invitro standardised

growth media

0.49 1.92 257.1 73.9

Cl. butyricum Minimised Growth

media

0.14 0.88 - -

Treated digested

agricultural

wastewater

0.24 1.36 110.9 17.9

Enriched Treated

digested agricultural

wastewater

0.38 1.75 279.4 31.9

[3]

Units Element Type Surface

area

(m2)

Material Total Cost

(USD, $)

Power

Usage

(MJ)

Steam

Usage

(kg/d)

Cooling

water

(kg/d)

Cross flow

microfiltration

unit

(waste

treatment)

Tanks Settling 319.83 Stainless steel

Type 304

77,799.50 -

1800

220000

Processing 201.56 75,593.64

Collection 201.56 75,593.64

Pumps Feed - Plastic/Metal 8,606.252 286.12

Recirculation - 8,606.25

Membrane Monolith tubular Ceramic 26,864.553 -

Heat

exchanger

Shell and tube 3.46 Stainless steel 10,555.98 -

Raw

Materials

Spent digester

effluents

- - -

Nutrient broth

preparation

unit

Tank Processing 201.56 Stainless steel

Type 304

75,593.64 -

1800

- Propeller

and baffles

Mixing 59.72 Stainless steel

Type 304

25,195.96 103.10

Raw

Materials

Powdered

chemicals i.e.

yeast extract,

glucose, sodium

chloride etc. and

tap water

Powder or

liquid

519,729.664 -

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

2 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 Waste to media conversion unit (cross flow microfiltration unit

)

Synthetic media preparation unit

Total plant direct cost (TPDC) (physical cost)

Equipment erection 0.4

0.7

0.2

0.1

none required

not applicable

provided in PCE

not applicable

none required

0.3

Piping

Instrumentation

Electricals

Buildings

Utilities

Storage

Site development

Ancillary buildings

Design and Engineering

Variable Costs

Raw materials 0 $519,729.66

Miscellaneous materials 0 $1,114.19

Utilities Cost

Steam $16,289.57 $16,289.57

Cooling water $6,787.32 -

Power $3,884.08 $1,111,37

Water $6,787.32 $27,182.98

Shipping & Packaging not applicable

Fixed Costs

Maintenance $27,794.74 $11,141.91

Operating labour $88,020.00 $88,020.00

Plant overheads $44,010.00 $44,010.00

Capital charges $72,266.33 $28,968.96

Insurance $5,558.95 $2,228.38

Local taxes not applicable

none required

not applicable

not applicable

not applicable

Royalties

Sales expenses

General overheads

R&D

Total annual production rate(rounded) $271,398.31 $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]

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

agricultural wastewater()

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35

Bio

mass

(g/L

)

Time (h)

In vitro optimised liquid mediaMinimised liquid mediaTreated agricultural wastewaterEnriched treated agricultural wastewater

[3]

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

3

6

15

A

B

1

2

E-23

14

8

5

7

9

10

11

12

13

16

17

18

Permeate

[4]

3

1

2

4

5

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

Permeate