CHEMICAL ENGINEERING TRANSACTIONS  
 

VOL. 70, 2018 

A publication of 

 

The Italian Association 
of Chemical Engineering 
Online at www.aidic.it/cet 

Guest Editors: Timothy G. Walmsley, Petar S. Varbanov, Rongxin Su, Jiří J. Klemeš 
Copyright © 2018, AIDIC Servizi S.r.l. 

ISBN 978-88-95608-67-9; ISSN 2283-9216 

Biological Remediation and Removal of Radioactive Metals 

and Complex Aromatic Compounds from Nuclear and 

Radioactive Waste 

Evans M. N. Chirwa*, Pulane E. Molokwane, Phumza V. Tikilili, Stanford S. 

Makgato, Emomotimi E. Bamuza-Pemu, Lindelwa Jay 

Water Utilisation and Environmental Engineering Division, Department of Chemical Engineering, University of Pretoria, 

Pretoria 0002, South Africa  

evans.chirwa@up.ac.za 

In pursuing carbon emission free technologies in the energy industry, interest in nuclear energy has grown. 

However, technology for handling radioactive waste containing organic materials are lacking. Methods are 

required for biodegradation of often recalcitrant irradiated organic compounds which are persistent in radioactive 

waste from the nuclear industry. In this study, a bioseparation process for extraction of 14C and de-radiation of 

nuclear graphite was investigated in a continuous flow sequencing-batch/biofilm reactor. Dehalogenation and 

mineralisation of aromatic halogenated compounds in the waste stream of a pebble bed manufacturing process 

was simulated by the photocatalytic/biological hybrid system in the 50 to 1,000 4-CP mg.L-1. The efficiency of 

degradation of the irradiated compounds was compared to conventional degradation using PAH degrading 

microorganisms. The Pathway of degradation was established for both systems by evaluating the intermediates 

of degradation of radiolabelled phenolic compounds with ortho-13C-ortho and para-13C phenol. Phenol 

degradation under photocatalytic conditions was shown to follow the catechol hydroquinone  

hydroxyhydroquinonebenzene-1,2,3-triol pathway, whereas the degradation in the biological stage followed 

the meta-cleavage pathway via catechol 2-hydromuconic semialdehyde pyruvate under aerobic conditions. 

This study demonstrates the potential of efficiently managing the organic component of nuclear waste using a 

cleaner, environmentally friendly biological process. 

1. Introduction 

The exponential increase in the world population in the last 200 years and the fast depletion of fossil fuel 

reserves has resulted in renewed interest in nuclear energy (Wang and Li, 2016). Additionally, the increased 

pressure to cut down on the burning of coal as a primary energy source due to its effects on global warming has 

caused many governments to re-open research into carbon free sources of energy such as solar, wind, nuclear 

and geothermal energy (McCauley, 2018). The main impeding factor in the implementation of nuclear energy is 

mainly due to the shortage of solutions for treatment and disposal of radioactive waste (Ismagilov et al., 2000).  

The buck of radioactive waste contains low and intermediate level waste (LLW, ILW) and a small amount of 

Transuranic (TU) elements. Low Level Waste (LLW) is generated from hospitals and industry, as well as the 

nuclear fuel cycle (Chirwa, 2011). LLW does not require shielding during handling and transport and is suitable 

for shallow land burial. To reduce its volume, it is often compacted or incinerated before disposal. The bulk of 

graphitic waste from Generation IV fast reactors could fall into this category. In this case, the limitation is space 

requirements rather than shielding and treatability.  

Wastewater from the general waste stream contains a large amount of organic pollutants. These radioactive 

organic compounds are recalcitrant and represent the highest risk to the environment through increase in total 

organic carbon (TOC) and toxicity. Physical and chemical treatment of organic aromatic pollutants has been 

shown to produce secondary radioactive waste which requires further treatment (IAEA, 2004). In this study, 

bioseparation of 14C from a 12C/14C nuclear graphite was attempted using locally isolated microorganisms. 

                                

 
 

 

 
   

                                                  
DOI: 10.3303/CET1870015 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Please cite this article as: Chirwa E.M.N., Molokwane P.E., Tikilili P.V., Makgato S.S., Bamuza-Pemu E.E., Jay L., 2018, Biological remediation 
and removal of radioactive metals and complex aromatic compounds from nuclear and radioactive waste , Chemical Engineering 
Transactions, 70, 85-90  DOI:10.3303/CET1870015   

85



Irradiated compounds were degraded in a photocatalytic/bioreactor hybrid system to achieve complete 

mineralisation of the compounds to CO2 and H2O.  

The study demonstrated that radiation tolerant consortia of bacteria can be isolated and optimised to efficiently 

degrade recalcitrant compounds under radiotoxic conditions. 

2. Configuration of the Generation IV Nuclear Reactor 

In order to free itself from reliance on coal as the principal source of energy, a country such as South Africa will 

need an energy source as dependable as coal while at the same time be able to remedy the potential 

environmental impacts of the chosen energy source. Nuclear energy has been discussed as the potential 

replacement of the coal generation capacity of South Africa. Nuclear energy offers a source with lower carbon 

emissions. However, it comes with the problem of radioactive waste which could create an environmental burden 

for thousands of years. Additionally, like coal, uranium on which nuclear energy is based, is a   

non-renewable resource on earth. The minimum requirement for new nuclear reactors is that they must be safe. 

Reactors with graphite as the neutron regular have been proposed. These reactors are able to self-regulate and 

shut down if the cooling agent – mostly a noble gas – is depleted. This reduces the risk of accidents such as 

those that occurred in Chernobyl (Ukraine) in 1986 and Tukushima (Japan) in 2011. One such technology is the 

Pebble Bed Modular Nuclear Reactor (PBMR). The PBMR is a Generation IV reactor capable of operating at 

very high temperatures (700 - 1,300 oC). The reactor is cooled by helium through the Brayton cycle (Figure 1a) 

(PBMR Pty Ltd, 2008). The core is filled with graphite pebbles the size of golf balls containing uranium kernels 

as the reactive component. The flux of neutrons is moderated by the TRISO oxide layer surrounding each fuel 

kernel and the graphite matrix surrounding the kernels (Figure 1b). 

 

Figure 1: (a) Schematic layout of the PBMR system and (b) anatomy of the pebble [Courtesy, PBMR (PTY) Ltd, 

SA]. 

 

 
 

 

 

 

 
 

 

 

 

 
Core 
 

Generator 

Low Pressure 
Turbine 

High Pressure 
Turbine 

Power Turbine 

Intercooler Pre-cooler 

High Pressure 

Compressor 
Low Pressure 
Compressor 

Helium Injection and 
removal from HICS 

Helium injection from 
HICS 

Recuperator 

(a) 

(b) 

High pressure 

compressor 

Low pressure 

compressor 

86



3. Carbon-14 study 

Biomass has long been suspected of being capable of preferential sequestration of the carbon atom based on 

mass. Fossils from the earlier earth suggest higher concentrations of 13C concentration in plants in an 

atmosphere with both 13C and 12C (Yim and Caron, 2006). This phenomenon is not evident in the modern plants; 

the presence of this capability in microorganisms from extreme environments may be likely due to preservation 

of archaic mechanisms in these organisms.  

In this study, a consortium of organisms was isolated from oil contaminated sites and radioactive wastewater 

storage ponds from radioisotope processing facilities in South Africa (Oboirien and Chirwa, 2007). Cultures from 

the different sources were characterised using the standard 16S rRNA genotype fingerprinting of sequenced 

DNA and DNA seprated by DGGE of amplified DNA. Initial analysis showed the presence of Klebsiella 

oxyoetica, Ponteae agglomerans, Klebsiella stutseli and Pseudomonas putida as the predominant active agents 

in the wastewater and sludge sources. K oxyoetica, K stutseli and P agglomerans showed resistance to radiation 

from nuclear graphite at levels up to 1.055  0.067 BqL-1, enough to inhibit metabolic activity in most mesophilic 

bacteria from the natural environment (Kudryasheva et al., 2008).  

The study was conducted in a continuously mixed reactor (growth vessel) constructed from Pyrex panels (30 

cm  20 cm  15 cm) with finely ground graphite as the principal carbon source (1,500 - 2,000 mg.L-1) and spikes 

of glucose (500 mg.L-1 every 48 h). The growth vessel was connected to a biofilter unit by submersible pump 

where particulate matter was isolated for further analysis as described earlier by Molokwane and Chirwa (2007) 

(Figure 2). The pump was also used for recirculation and mixing.  

 

Figure 2: Experimental setup for bioseparation of 14C from 12C/14C nuclear graphite (Adopted from Molokwane 

and Chirwa, 2007). 

The samples collected from the aeration chamber (Sample A) and PVC trays (Sample B) were analysed for 14C 

content by the Tri-Carb 2900TR Liquid Scintillator (Perkin-Elmer, Shelton, Connecticut, USA). The results from 

detailed analysis after operating the bioreactor for 5 days after adding 14C containing graphite showed a higher 
14C count in Sample B from the biofilter racks where the bacteria cells were mostly concentrated (Table 1).  

Table 1: Results of the C-14 bioseparation experimental in the fed-batch continuous flow mixed reactor after 

operation for 5 days. 

 Sample A  
(from reservoir) 

Sample B  
(from biofilter) 

CO2 measurement (torr)   
Filter 254 171.8 

Bacteria 80 338.2 
Filler 176 0 
Total 510 510 

14C measurement   
Detection time (min) 940 1,124 

Total counts 5,157 10,818 
Sample Activity (cpm) 5.48 11.51 

 

Biofilter with 
PVC Sieves 

Liquid 

Sample 

Stirrer 

Growth Vessel 

Submersible 
Pump 

Sample 

Sample 

87



4. Photocatalytic degradation pathway 

The degradation pathway in non-radioactive phenolic sample during a photocatalytic process showed than 

phenol mineralised in a stepwise process with organic intermediate observed at different times until all 

compounds were degraded (Figure 3) (Chirwa and Bamuza-Pemu, 2010). For higher radioisotopes of C, the 

 

Figure 3: Chromatograms showing intermediates formed at different times during the photocatalytic degradation 

of mixture of Phenol, 2-CP, 4-CP,and 4-NP.  

process was evaluated using 13C-ortho and 13C-para phenol and it showed that the degradation pathway 

followed the oxidative process involving the intermediate formation catechol  hydroquinone  

hydroxyhydroquinone  benzene-1,2,3-triol (Figure 4). These results suggest that, for most of the irradiate 

compounds, accumulation of intermediate less toxic compounds could benefit the microbial cultures that could 

be used in the second stage after initial exposure to photocatalytic degradation.   

5. Biological degradation pathway 

In nuclear fuel processing, the problem is the generation of large volumes of partially water soluble wastes that 

often contain toxic organic compounds. The use of decontamination reagents such as carbon tetrachloride 

(CCl4) together with phenolic tar results in wastewater with a high content of chlorophenols. Chlorophenols 

cause serious environmental concerns because of their toxic and carcinogenic effects on living organisms 

(Olaniran and Igbinosa, 2011). Overall, there are 19 possible configurations of chlorinated phenols in nature. 

One of these, 4-chlorophenol (4-CP), represents the most basic structure and its degradation pathway has been 

extensively studied in several organisms (Bian et al., 2011). 4-CP was thus used as a model compound 

representing in the evaluation of the biological degradation of recalcitrant aromatic pollutants in heavily polluted 

wastewaters such as effluents from nuclear fuel processing and radioisotope manufacturing plants.  

The dehalogenation of the phenolic compound happened rather quickly as shown in the photo-oxidation process 

(Figure 4). The biological degradation of the remnant compound will follow the meta-cleavage pathway under 

aerobic conditions or the ortho-cleavage pathway under anaerobic conditions. When the conditions are 

transitional, a truncated meta-cleavage pathway exists in which phenol is converted to HMSA followed by 

conversion to Acetate and Succinic acid. Evidence of the existence was of the truncated meta-cleavage pathway 

 

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88



was demonstrated earlier by Nkhalambayausi-Chirwa and Wang (2001) in reaction where the final products 

were acetate and succinate in an oxygen-stressed biofilm environment (Figure 5). 

 

 

Figure 4: The pathway tracking of degradation products during photolytic degradation of radiocarbon labelled 
13C phenol using GC-MS analysis. 

 

Figure 5: Chromatograms showing the degradation and mineralisation levels during photolytic degradation and 

biodegradation in a CSTR as the second stage. 

 

 

 

 
 

 

 

Min 

Min 

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89



6. Conclusion 

For the first, the bioseparation of 14C from a 14C/12C graphitic matrix was demonstrated. The microbial 

consortium that achieved the bioseparation process contained some archaebacterial Panteae agglomerans and 

Klebsiella stutseli species. Degradation of aromatic compounds was most probably facilitated by 

Pseaudomansa putida serotypes. The processes investigates show the potential of lowering radiotoxicity of 

evolved irradiated waste followed by mineralisation of complex aromatic organic compounds using a two-stage 

photocatalytic-biological process.  

Acknowledgments 

We thank PBMR and NRF-THRIP for funding this research. We especially would like to thank PBMR for 

permission to use results from the preliminary study on 14C removal conducted at PBMR. Research on the 

degradation of aromatic compounds was funded by National Research Foundation of South Africa (NRF) 

through Grant No. FA2007030400002 awarded to Prof Evans M. N. Chirwa at the University of Pretoria and 

SANHARP through a Master's and PhD bursary awarded to members of the research group. 

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